Efficacy of anti-trop-2-sn-38 antibody drug conjugates for therapy of tumors relapsed/refractory to checkpoint inhibitors

ABSTRACT

The present invention relates to therapeutic ADCs comprising SN-38 attached to an anti-Trop-2 antibody or antigen-binding antibody fragment, more particularly sacituzumab govitecan. The ADC is administered to a subject with a Trop-2 positive cancer that is resistant to or relapsed from prior treatment with a checkpoint inhibitor. The therapy is effective to treat cancers that are resistant to checkpoint inhibitors.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/490,535, filed Apr. 18, 2017, which claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application 62/328,289, filedApr. 27, 2016, the entire text of which is incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2017, isnamed IMM366US2_SL.txt. and is 13,002 bytes in size.

FIELD OF THE INVENTION

The present invention relates to therapeutic use of antibody-drugconjugates (ADCs) comprising anti-Trop-2 antibodies or antigen-bindingfragments thereof and camptothecins, such as SN-38, with improvedability to target various cancer cells in human subjects. In preferredembodiments, the antibodies and therapeutic moieties are linked via anintracellularly-cleavable linkage that increases therapeutic efficacy.In more preferred embodiments, the ADCs are administered at specificdosages and/or specific schedules of administration that optimize thetherapeutic effect. Surprisingly, the optimized dosages and schedules ofadministration of SN-38-conjugated antibodies, such as sacituzumabgovitecan, show unexpected superior efficacy that could not have beenpredicted from animal model studies, allowing effective treatment ofcancers that are resistant to checkpoint inhibitor antibodies such asatezolizumab, pembrolizumab, nivolumab, pidilizumab, durvalumab,atezolizumab, ipilimumab or tremelimumab. In specific embodiments, theADC may be administered to a human subject with a Trop-2 positive cancerat a dosage of between 3 and 18 mg/kg, more preferably between 4 and 12mg/kg, most preferably between 8 and 10 mg/kg. Surprisingly, theanti-Trop-2-SN38 antibody drug conjugates (ADCs) are effective to treatTrop-2 positive cancers in patients who had relapsed from or shownresistance to checkpoint inhibitor therapy, such as pancreatic cancer,triple-negative breast cancer, small cell lung cancer, endometrialcancer, urothelial cancer and non-small cell lung cancer.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer in humans, andvirtually no application in other diseases, such as infectious andautoimmune diseases. The toxic agent is most commonly a chemotherapeuticdrug, although particle-emitting radionuclides, or bacterial or planttoxins, have also been conjugated to MAbs, especially for the therapy ofcancer (Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3): 193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

A need exists for more effective methods of preparing and administeringantibody-CPT conjugates, such as anti-Trop-2-SN-38 conjugates (e.g.,sacituzumab govitecan). Preferably, the methods comprise optimizeddosing and administration schedules that maximize efficacy and minimizetoxicity, for use in patients with Trop-2 positive cancers that arerelapsed from or resistant to checkpoint inhibitor antibodies.

SUMMARY OF THE INVENTION

As used herein, the abbreviation “CPT” may refer to camptothecin or anyof its derivatives, such as SN-38, unless expressly stated otherwise.The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparing andadministering CPT-antibody ADCs. Preferably, the camptothecin is SN-38and the antibody is an anti-Trop-2 antibody (e.g., sacituzumabgovitecan). The disclosed methods and compositions are of use for thetreatment of diseases which are refractory or less responsive to otherforms of therapy, such as Trop-2+ cancers that are resistant to orrelapsed from treatment with checkpoint inhibitor antibodies.

Preferably, the antibody moiety of the ADC is a monoclonal antibody,antibody fragment, bispecific or other multivalent antibody, or otherantibody-based molecule or compound. The antibody can be of variousisotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferablycomprising human IgG1 hinge and constant region sequences. The antibodyor fragment thereof can be a chimeric human-mouse, a chimerichuman-primate, a humanized (human framework and murine hypervariable(CDR) regions), or fully human antibody, as well as variations thereof,such as half-IgG4 antibodies (referred to as “unibodies”), as describedby van der Neut Kolfschoten et al. (Science 2007; 317:1554-1557). Morepreferably, the antibody or fragment thereof may be designed or selectedto comprise human constant region sequences that belong to specificallotypes, which may result in reduced immunogenicity when the ADC isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

An exemplary anti-Trop-2 antibody is a humanized RS7 (hRS7) antibody,comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ IDNO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3)and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).

In a preferred embodiment, the chemotherapeutic moiety is selected fromcamptothecin (CPT) and its analogs and derivatives and is morepreferably SN-38. However, other chemotherapeutic moieties that may beutilized include taxanes (e.g, baccatin III, taxol), epothilones,anthracyclines (e.g., doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX) or a prodrug formof 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series574, published by American Chemical Society, Washington D.C., 1995(332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al.,Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest. NewDrugs 17:361-373, 1999), and the like. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 ormore chemotherapeutic moieties, most preferably about 6 to about 12chemotherapeutic moieties.

Various embodiments may concern use of the subject methods andcompositions to treat cancers that express human Trop-2, including butnot limited to carcinomas such as carcinomas of the esophagus, pancreas,lung, stomach, colon and rectum, urinary bladder, breast, ovary, uterus,kidney, urothelium and prostate.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, full doses of each can also be given. Inalternative embodiments, the ADCs may be administered in combinationwith an interferon, a checkpoint inhibitor antibody, a Bruton tyrosinekinase inhibitor, a PI3K inhibitor, a PARP inhibitor or a microtubuleinhibitor, as discussed below.

Preferred optimal dosing of ADCs may include an i.v. dosage of between 3mg/kg and 18 mg/kg, preferably given either weekly, twice weekly orevery other week. The optimal dosing schedule may include treatmentcycles of two consecutive weeks of therapy followed by one, two, threeor four weeks of rest, or alternating weeks of therapy and rest, or oneweek of therapy followed by two, three or four weeks of rest, or threeweeks of therapy followed by one, two, three or four weeks of rest, orfour weeks of therapy followed by one, two, three or four weeks of rest,or five weeks of therapy followed by one, two, three, four or five weeksof rest, or administration once every two weeks, once every three weeksor once a month. Treatment may be extended for any number of cycles,preferably at least 2, at least 4, at least 6, at least 8, at least 10,at least 12, at least 14, or at least 16 cycles. Exemplary dosages ofuse may include 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, and 18 mg/kg. Preferred dosages are 4, 6, 8, 9, 10, or 12 mg/kg.

In certain alternative embodiments, where the ADC is administeredsubcutaneously, dosages of ADCs such as sacituzumab govitecan (IMMU-132)may be limited by the ability to concentrate the ADC withoutprecipitation or aggregation, as well as the volume of administrationthat may be given subcutaneously (preferably, 1, 2, or 3 ml or less).Consequently, for subcutaneous administration the ADC may be given at1.5 to 4 mg/kg, given daily for 1 week, or 3 times weekly for 2 weeks,or twice weekly for two weeks, followed by rest. Where multiple sites ofs.c. injection are used, dosages of up to 8 mg/kg may be provided.Maintenance doses of ADC may be administered i.v. or s.c. every two tothree weeks or monthly after induction. Alternatively, induction mayoccur with two to four cycles of i.v. administration at 8-10 mg/kg (eachcycle with ADC administration on Days 1 and 8 of two 21-day cycles witha one-week rest period in between), followed by s.c. administration asactive dosing one or more times weekly or as maintenance therapy. Dosingmay be adjusted based on interim tumor scans and/or by analysis ofTrop-2 positive circulating tumor cells.

The person of ordinary skill will realize that a variety of factors,such as age, general health, specific organ function or weight, as wellas effects of prior therapy on specific organ systems (e.g., bonemarrow) may be considered in selecting an optimal dosage of ADC, andthat the dosage and/or frequency of administration may be increased ordecreased during the course of therapy. The dosage may be repeated asneeded, with evidence of tumor shrinkage observed after as few as 4 to 8doses. The optimized dosages and schedules of administration disclosedherein show unexpected superior efficacy and reduced toxicity in humansubjects, which could not have been predicted from animal model studies.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including checkpoint inhibitor therapy.

The subject methods may include use of CT and/or PET/CT, or MRI, tomeasure tumor response at regular intervals. Blood levels of tumormarkers, such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA 15.3,or PSA, may also be monitored. Dosages and/or administration schedulesmay be adjusted as needed, according to the results of imaging and/ormarker blood levels.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea and vomiting observed, or manageable neutropenia. A furthersurprising result is the lack of accumulation of the antibody-drugconjugate, unlike other products that have conjugated SN-38 to albumin,PEG or other carriers. The lack of accumulation is associated withimproved tolerability and lack of serious toxicity even after repeatedor increased dosing. These surprising results allow optimization ofdosage and delivery schedule, with unexpectedly high efficacies and lowtoxicities. The claimed methods provide for shrinkage of solid tumors,in individuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by longest diameter). The person of ordinaryskill will realize that tumor size may be measured by a variety ofdifferent techniques, such as total tumor volume, maximal tumor size inany dimension or a combination of size measurements in severaldimensions. This may be with standard radiological procedures, such ascomputed tomography, ultrasonography, and/or positron-emissiontomography. The means of measuring size is less important than observinga trend of decreasing tumor size with ADC treatment, preferablyresulting in elimination of the tumor.

While the ADC may be administered as a periodic bolus injection, inalternative embodiments the ADC may be administered by continuousinfusion of antibody-drug conjugates. In order to increase the Cmax andextend the PK of the ADC in the blood, a continuous infusion may beadministered for example by indwelling catheter. Such devices are knownin the art, such as HICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see,e.g., Skolnik et al., Ther Drug Monit 32:741-48, 2010) and any suchknown indwelling catheter may be used. A variety of continuous infusionpumps are also known in the art and any such known infusion pump may beused. The dosage range for continuous infusion may be between 0.1 and3.0 mg/kg per day. More preferably, these ADCs can be administered byintravenous infusions over relatively short periods of 2 to 5 hours,more preferably 2-3 hours.

In particularly preferred embodiments, the ADCs and dosing schedules maybe efficacious in patients resistant to standard therapies. For example,the anti-Trop-2 sacituzumab govitecan may be administered to a patientwho has not responded to prior therapy with irinotecan, the parent agentof SN-38. Surprisingly, the irinotecan-resistant patient may show apartial or even a complete response to sacituzumab govitecan. Theability of the ADC to specifically target the tumor tissue may overcometumor resistance by improved targeting and enhanced delivery of thetherapeutic agent. The ADCs may show similar improved efficacy and/ordecreased toxicity, compared to alternative standard therapeutictreatments such as checkpoint inhibitor antibodies.

A specific preferred subject may be a metastatic colorectal cancerpatient, metastatic pancreatic cancer patient, a triple-negative breastcancer patient, a HER+, ER+, progesterone+ breast cancer patient, ametastatic non-small-cell lung cancer (NSCLC) patient, a metastaticsmall-cell lung cancer patient, a metastatic stomach cancer patient, ametastatic renal cancer patient, a metastatic urothelial cancer patient,a metastatic urinary bladder cancer patient, a metastatic ovarian cancerpatient, or a metastatic uterine cancer patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Summary of CT scan results for patient with checkpoint inhibitorresistant metastatic TNBC, treated with 10 mg/kg sacituzumab govitecan.

FIG. 2. Baseline CT images for patient with checkpoint inhibitorresistant metastatic TNBC, showing axial images (top row) and sagittalimages (bottom row). Tumors are indicated by arrows.

FIG. 3. Comparison of CT scans for target lesions 1 and 2, before andafter treatment with 10 mg/kg sacituzumab govitecan (IMMU-132). Baselineimage is shown on top and second response assessment after sacituzumabgovitecan therapy is shown on the bottom. Shrinkage of tumors induced byIMMU-132 was clearly observed.

FIG. 4. Comparison of CT scans for target lesion 3, before and aftertreatment with 10 mg/kg sacituzumab govitecan (IMMU-132). Baseline imageis shown on top and second response assessment after sacituzumabgovitecan therapy is shown on the bottom. Shrinkage of tumor induced byIMMU-132 was clearly observed.

FIG. 5. Summary of results for patients with checkpoint inhibitorresistant tumors treated with IMMU-132.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317 (14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, immunoconjugates,drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radionuclides,oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents,chemotherapeutic agents, cyokines, chemokines, prodrugs, enzymes,binding proteins or peptides or combinations thereof.

An immunoconjugate is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent. Where the therapeutic agent is adrug, the resulting immunoconjugate is an antibody-drug conjugate orADC.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

CPT is an abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin, such as SN-38. The structures ofcamptothecin and some of its analogs, with the numbering indicated andthe rings labeled with letters A-E, are given in formula 1 in Chart 1below.

Chart 1

Anti-Trop-2 Antibodies

Various embodiments concern use of antibodies or fragments thereof thatbind to Trop-2. In a specific preferred embodiment, the anti-Trop-2antibody may be a humanized RS7 antibody (see, e.g., U.S. Pat. No.7,238,785, incorporated herein by reference in its entirety), comprisingthe light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2(SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavychain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG,SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). The amino acid sequenceof human Trop-2 is publicly known (see, e.g., NCBI Accession No.P09758.3). In addition to its role as a tumor-associated calcium signaltransducer (Ripani et al., Int J Cancer 1998; 76:671-6), the expressionof human Trop-2 was shown to be necessary for tumorigenesis andinvasiveness of colon cancer cells, which could be effectively reducedwith a polyclonal antibody against the extracellular domain of Trop-2(Wang et al., Mol Cancer Ther 2008; 7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful for ADCproduction. (Id.) It is well documented that internalization of ADCs isa requirement for anti-tumor activity. (Pastan et al., Cell 47:641,1986) Internalization of drug ADCs has been described as a major factorin anti-tumor efficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85:1189, 1988) Thus, the RS7 antibody exhibits several important propertiesfor therapeutic applications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. ADC PF 06263507,comprising an anti-5T4 (anti-Trop-2) antibody attached to the tubulininhibitor monomethylauristatin F (MMAF) is available from Pfizer, Inc.(Groton, Conn.) (see, e.g., Sapra et al., 2013, Mol Cancer Ther12:38-47). U.S. Pat. No. 8,715,662 discloses anti-Trop-2 antibodiesproduced by hybridomas deposited at the AID-ICLC (Genoa, Italy) withdeposit numbers PD 08019, PD 08020 and PD 08021. U.S. Patent ApplicationPubl. No. 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 andKM4590. U.S. Pat. No. 8,309,094 (Wyeth) discloses antibodies A1 and A3,identified by sequence listing. The Examples section of each patent orpatent application cited above in this paragraph is incorporated hereinby reference. Non-patent publication Lipinski et al. (1981, Proc Natl.Acad Sci USA, 78:5147-50) disclosed anti-Trop-2 antibodies 162-25.3 and162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. Methods for producing humanized,human or chimeric antibodies were also known. The person of ordinaryskill, reading the instant disclosure in light of general knowledge inthe art, would have been able to make and use the genus of anti-Trop-2antibodies in the subject ADCs.

Use of antibodies against targets related to Trop-2 has been disclosedfor immunotherapeutics other than ADCs. The murine anti-Trop-1 IgG2aantibody edrecolomab (PANOREX®) has been used for treatment ofcolorectal cancer, although the murine antibody is not well suited forhuman clinical use (Baeuerle & Gires, 2007, Br. J Cancer 96:417-423).Low-dose subcutaneous administration of ecrecolomab was reported toinduce humoral immune responses against the vaccine antigen (Baeuerle &Gires, 2007). Adecatumumab (MT201), a fully human anti-Trop-1 antibody,has been used in metastatic breast cancer and early-stage prostatecancer and is reported to act through ADCC and CDC activity (Baeuerle &Gires, 2007). MT110, a single-chain anti-Trop-1/anti-CD3 bispecificantibody construct has reported efficacy against ovarian cancer(Baeuerle & Gires, 2007). Proxinium, an immunotoxin comprisinganti-Trop-1 single-chain antibody fused to Pseudomonas exotoxin, hasbeen tested in head-and-neck and bladder cancer (Baeuerle & Gires,2007). None of these studies contained any disclosure of the use ofanti-Trop-2 ADCs, particularly in patients resistant to treatment withcheckpoint inhibitor antibodies.

Camptothecin Conjugates

Non-limiting methods and compositions for preparing ADCs comprising acamptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide (oracetylene) moiety of said defined bifunctional PEG is optionallyattached to the N-terminus of an L-amino acid or a polypeptide, with theC-terminus attached to the amino group of amino alcohol, and the hydroxygroup of the latter is attached to the hydroxyl group of the drug in theform of carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these are deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group is readilydeprotected under physiological pH conditions after the bioconjugate isadministered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. Click chemistry takes place inaqueous solution at near-neutral pH conditions, and is thus amenable fordrug conjugation. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction.

While the present application focuses on use of antibodies or antibodyfragments as targeting moieties, the skilled artisan will realize thatwhere a conjugate comprises an antibody or antibody fragment, anothertype of targeting moiety, such as an aptamer, avimer, affibody orpeptide ligand, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,

MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (2)

where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the conjugate of the preferred embodiment of formula 2,m is 0, A′ is L-valinol, and the drug is exemplified by SN-38. Theresultant structure is shown in formula 3.

In another example of the conjugate of the preferred embodiment offormula 2, m is 1 and represented by a derivatized L-lysine, A′ isL-valinol, and the drug is exemplified by SN-38. The structure is shownin formula 4.

In this embodiment, an amide bond is first formed between the carboxylicacid of an amino acid such as lysine and the amino group of valinol,using orthogonal protecting groups for the lysine amino groups. Theprotecting group on the N-terminus of lysine is removed, keeping theprotecting group on the side chain of lysine intact, and the N-terminusis coupled to the carboxyl group on the defined PEG with azide (oracetylene) at the other end. The hydroxyl group of valinol is thenattached to the 20-chloroformate derivative of 10-hydroxy-protectedSN-38, and this intermediate is coupled to an L2 component carrying theantibody-binding moiety as well as the complementary acetylene (orazide) group involved in the click cycloaddition chemistry. Finally,removal of protecting groups at both lysine side chain and SN-38 givesthe product of this example, shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-38 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 2 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 5, referred to asMAb-CLX-SN-38). Single amino acid of AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C1-C10 alkyl groups.

A particularly preferred embodiment of MAb-CLX-SN-38 of formula 5,wherein the single amino acid AA is L-lysine and R═H, and the drug isexemplified by SN-38 (formula 6; referred to as MAb-CL2A-SN-38). Thestructure differs from the linker MAb-CL2-SN-38 in the substitution of asingle lysine residue for a Phe-Lys dipeptide found in the CL2 linker.The Phe-Lys dipeptide was designed as a cathepsin B cleavage site forlysosomal enzyme, which was considered to be important for intracellularrelease of bound drug. Surprisingly, despite the elimination of thecathepsin-cleavage site, ADCs comprising a CL2A linker are at least asefficacious, and may be more efficacious in vivo than those comprising aCL2 linker.

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine. An example of thisembodiment is represented by the formula ‘7’ below, wherein the phenolichydroxyl group of SN-38 is derivatized as a carbamate with a substitutedethylenediamine, with the other amine of the diamine derivatized as acarbamate with a 4-aminobenzyl alcohol, and the latter's amino group isattached to Phe-Lys dipeptide. In this structure (formula 7), R and R′are independently hydrogen or methyl. It is referred to asMAb-CL17-SN-38 or MAb-CL2E-SN-38, when R═R′=methyl.

In certain embodiments, AA comprises a polypeptide moiety, preferably adi, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO: 20)(Trouet et al., 1982). Another example is a Phe-Lys moiety that iscleavable by lysosomal cathepsin.

In a preferred embodiment, the L1 component of the conjugate contains adefined polyethyleneglycol (PEG) spacer with 1-30 repeating monomericunits. In a further preferred embodiment, PEG is a defined PEG with 1-12repeating monomeric units. The introduction of PEG may involve usingheterobifunctionalized PEG derivatives which are available commercially.The heterobifunctional PEG may contain an azide or acetylene group. Anexample of a heterobifunctional defined PEG containing 8 repeatingmonomeric units, with ‘NHS’ being succinimidyl, is given below informula 8:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single antibody-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single antibody-binding moiety is shown below. The ‘L2’component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. In a morepreferred embodiment, attachment of SN-38 to reduced disulfidesulfhydryl groups results in formation of an antibody-SN-38 ADC with 6SN-38 moieties covalently attached per antibody molecule. Other methodsof providing cysteine residues for attachment of drugs or othertherapeutic agents are known, such as the use of cysteine engineeredantibodies (see U.S. Pat. No. 7,521,541, the Examples section of whichis incorporated herein by reference.)

In alternative preferred embodiments, the chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), Pro-2PDOX, CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin,ansamycins, and epothilones. In a more preferred embodiment, thechemotherapeutic moiety is SN-38. Preferably, in the conjugates of thepreferred embodiments, the antibody links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; most preferably about 6 to about 12 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L2’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of this work, a process was surprisingly discovered bywhich CPT drug-linkers can be prepared wherein CPT additionally has a10-hydroxyl group. This process involves, but is not limited to, theprotection of the 10-hydroxyl group as a t-butyloxycarbonyl (BOC)derivative, followed by the preparation of the penultimate intermediateof the drug-linker conjugate. Usually, removal of BOC group requirestreatment with strong acid such as trifluoroacetic acid (TFA). Underthese conditions, the CPT 20-O-linker carbonate, containing protectinggroups to be removed, is also susceptible to cleavage, thereby givingrise to unmodified CPT. In fact, the rationale for using a mildlyremovable methoxytrityl (MMT) protecting group for the lysine side chainof the linker molecule, as enunciated in the art, was precisely to avoidthis possibility (Walker et al., 2002). It was discovered that selectiveremoval of phenolic BOC protecting group is possible by carrying outreactions for short durations, optimally 3-to-5 minutes. Under theseconditions, the predominant product was that in which the ‘BOC’ at10-hydroxyl position was removed, while the carbonate at ‘20’ positionwas intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The 10-hydroxy protecting group, which convertsthe 10-OH into a phenolic carbonate or a phenolic ester, is readilydeprotected by physiological pH conditions or by esterases after in vivoadministration of the conjugate. The faster removal of a phenoliccarbonate at the 10 position vs. a tertiary carbonate at the 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 1-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or a simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it can be an alkoxymoiety such as “CH₃—(CH₂)n-O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody (indicated herein as “MAb”).

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the antibody is a monoclonal antibody (MAb). In otherembodiments, the antibody may be a multivalent and/or multispecific MAb.The antibody may be a murine, chimeric, humanized, or human monoclonalantibody, and said antibody may be in intact, fragment (Fab, Fab′,F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form, or ofan IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.

In a preferred embodiment, the antibody binds to an antigen or epitopeof an antigen expressed on a cancer or malignant cell, most preferablyTrop-2. The cancer cell is preferably a cell from a hematopoietic tumor,carcinoma, sarcoma, melanoma or a glial tumor. A preferred malignancy tobe treated according to the present invention is a malignant solid tumoror hematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof.

Checkpoint Inhibitor Antibodies

Studies with checkpoint inhibitor antibodies for cancer therapy havegenerated unprecedented response rates in cancers previously thought tobe resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013,Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85; Pardoll, 2012, Nature Reviews 12:252-264; Mavilio & Lugli,).Therapy with antagonistic checkpoint blocking antibodies against CTLA-4,PD-1 and PD-L1 are one of the most promising new avenues ofimmunotherapy for cancer and other diseases. In contrast to the majorityof anti-cancer agents, checkpoint inhibitor do not target tumor cellsdirectly, but rather target lymphocyte receptors or their ligands inorder to enhance the endogenous antitumor activity of the immune system.(Pardoll, 2012, Nature Reviews 12:252-264) Because such antibodies actprimarily by regulating the immune response to diseased cells, tissuesor pathogens, they may be used in combination with other therapeuticmodalities, such as antibody-drug conjugates (ADCs), to enhance theanti-tumor effect of the ADCs.

Programmed cell death protein 1 (PD-1, also known as CD279) encodes acell surface membrane protein of the immunoglobulin superfamily, whichis expressed in B cells and NK cells (Shinohara et al., 1995, Genomics23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45;Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews12:252-264). Anti-PD1 antibodies have been used for treatment ofmelanoma, non-small-cell lung cancer, bladder cancer, prostate cancer,colorectal cancer, head and neck cancer, triple-negative breast cancer,leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N EnglJ Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Bergeret al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013,Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85).

Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, MERCK),nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011,CURETECH LTD.). Anti-PD1 antibodies are commercially available, forexample from ABCAM® (AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) andAFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).

Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is aligand for PD-1, found on activated T cells, B cells, myeloid cells andmacrophages. The complex of PD-1 and PD-L1 inhibits proliferation ofCD8+ T cells and reduces the immune response (Topalian et al., 2012, NEngl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).Anti-PDL1 antibodies have been used for treatment of non-small cell lungcancer, melanoma, colorectal cancer, renal-cell cancer, pancreaticcancer, gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013,Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51).

Exemplary anti-PDL1 antibodies include MDX-1105 (MEDAREX), durvalumab(MEDI4736, MEDIMMUNE) atezolizumab (TECENTRIQ®, MPDL3280A, GENENTECH)and BMS-936559 (BRISTOL-MYERS SQUIBB). Anti-PDL1 antibodies are alsocommercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH1).

Cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152) is also amember of the immunoglobulin superfamily that is expressed exclusivelyon T-cells. CTLA-4 acts to inhibit T cell activation and is reported toinhibit helper T cell activity and enhance regulatory T cellimmunosuppressive activity (Pardoll, 2012, Nature Reviews 12:252-264).Anti-CTL4A antibodies have been used in clinical trials for treatment ofmelanoma, prostate cancer, small cell lung cancer, non-small cell lungcancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al.,2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wadaet al., 2013, J Transl Med 11:89).

Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-MyersSquibb) and tremelimumab (PFIZER). Anti-PD1 antibodies are commerciallyavailable, for example from ABCAM® (AB134090), SINO BIOLOGICAL INC.(11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572,PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recentlyreceived FDA approval for treatment of metastatic melanoma (Wada et al.,2013, J Transl Med 11:89).

These and other known checkpoint inhibitor antibodies may be used incombination with IMMU-132. In preferred embodiments, IMMU-132, alone orin combination, is effective to treat patients with cancers refractoryto checkpoint inhibitor antibodies alone.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. The person of ordinary skill will realize that whereantibodies are to be administered to human subjects, the antibodies willbind to human antigens.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953;5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.), in which the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The transgenic mice were transformed with germline-configured YACs(yeast artificial chromosomes) that contained portions of the human IgHand Ig kappa loci, including the majority of the variable regionsequences, along accessory genes and regulatory sequences. The humanvariable region repertoire may be used to generate antibody producing Bcells, which may be processed into hybridomas by known techniques. AXENOMOUSE® immunized with a target antigen will produce human antibodiesby the normal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of geneticallyengineered mice are available, each of which is capable of producing adifferent class of antibody. Transgenically produced human antibodieshave been shown to have therapeutic potential, while retaining thepharmacokinetic properties of normal human antibodies (Green et al.,1999). The skilled artisan will realize that the claimed compositionsand methods are not limited to use of the XENOMOUSE® system but mayutilize any transgenic animal that has been genetically engineered toproduce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. Nos. 4,036,945; 4,331,647; Nisonoff et al., 1960, Arch. Biochem.Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al.,1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan etal. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. More preferably, the antibodies internalize rapidlyfollowing binding. An exemplary rapidly internalizing antibody is theLL1 (anti-CD74) antibody, with a rate of internalization ofapproximately 8×10⁶ antibody molecules per cell per day (e.g., Hansen etal., 1996, Biochem J. 320:293-300). Thus, a “rapidly internalizing”antibody may be one with an internalization rate of about 1×10⁶ to about1×10⁷ antibody molecules per cell per day. Antibodies of use in theclaimed compositions and methods may include MAbs with properties asrecited above. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 orKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, alsoknown as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections. In a particularly preferred embodiment, the antibodyis hRS7. The person of ordinary skill will realize that in certainembodiments, antibodies against other TAAs besides Trop-2 may be used incombination with an anti-Trop-2 antibody.

Other useful antigens that may be targeted include carbonic anhydraseIX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20,1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38,CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70,CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154,CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP), VEGF (e.g.,bevacizumab, fibronectin splice variant), ED-B fibronectin (e.g., L19),EGP-1 (TROP-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1) (e.g.,cetuximab), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor, Ga733, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24, HER-2/neu,insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides,HCG, the HLA-DR antigen to which L243 binds, CD66 antigens, i.e.,CD66a-d or a combination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B,macrophage migration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4,MUC5ac, placental growth factor (PlGF), PSA (prostate-specific antigen),PSMA, PAM4 antigen, PD-1 receptor, NCA-95, NCA-90, A3, A33, Ep-CAM,KS-1, Le(y), mesothelin, 5100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), TROP-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaet al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8). Another useful target for breast cancer therapy is theLIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).The CD47 antigen is a further useful target for cancer stem cells (see,e.g., Naujokat et al., 2014, Immunotherapy 6:290-308; Goto et al., 2014,Eur J Cancer 50:1836-46; Unanue, 2013, Proc Natl Acad Sci USA110:10886-7).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):55565-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3×anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3×anti-Trop-2,anti-CD3×anti-CD20, anti-CD3×anti-CD22, anti-CD3×anti-HLA-DR oranti-CD3×anti-CD74. In certain embodiments, the techniques andcompositions for ADC therapy disclosed herein may be used in combinationwith bispecific or multispecific antibodies. For example, ananti-Trop-2×anti-CD3 bsAb may be administered before, simultaneouslywith or after an anti-Trop-2 ADC.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos.4,946,778 and 5,132,405, the Examples section of each of which isincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens.

DOCK-AND-LOCK™ (DNL®)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111 and8,034,352, the Examples section of each of which is incorporated hereinby reference.) Generally, the technique takes advantage of the specificand high-affinity binding interactions that occur between a dimerizationand docking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has □ and □ isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RI□, RI□, RII□ andRII□. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRII□, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRII□ are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL™ complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 7) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 8) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 9) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 10)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RII□□ isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RI□□ form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 11) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 12) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEO ID NO: 13) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 14)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEE AK PKA RIβ(SEQ ID NO: 15) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILAPKA RIIα (SEQ ID NO: 16) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 17) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:18) and veltuzumab (SEQ IDNO:19).

Rituximab heavy chain variable region sequence (SEQ ID NO:18)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Veltuzumab heavy chain variable region (SEQ ID NO:19)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 1compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 1, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete allotype 214 (allotype) 356/358 (allotype)431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R 3 E/M— A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. Nos. 5,475,096 and5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from Affibody AB(Solna, Sweden). Affibodies are small proteins that function as antibodymimetics and are of use in binding target molecules. Affibodies weredeveloped by combinatorial engineering on an alpha helical proteinscaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et al., 1997, NatBiotechnol 15:772-77). The affibody design is based on a three helixbundle structure comprising the IgG binding domain of protein A (Nord etal., 1995; 1997). Affibodies with a wide range of binding affinities maybe produced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.).

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that affibodies or fynomers may be usedas targeting molecules in the practice of the claimed methods andcompositions.

Immunoconjugates

In certain embodiments, a cytotoxic drug or other therapeutic ordiagnostic agent may be covalently attached to an antibody or antibodyfragment to form an immunoconjugate. In some embodiments, a drug orother agent may be attached to an antibody or fragment thereof via acarrier moiety. Carrier moieties may be attached, for example to reducedSH groups and/or to carbohydrate side chains. A carrier moiety can beattached at the hinge region of a reduced antibody component viadisulfide bond formation. Alternatively, such agents can be attachedusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of the ADC is anantibody fragment. However, it is possible to introduce a carbohydratemoiety into the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section ofwhich is incorporated herein by reference. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for ADC formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of ADC formation are disclosed, for example,in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338; 5,677,427; 5,697,902;5,716,595; 6,071,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104;6,962,702; 7,033,572; 7,147,856; and 7,259,240, the Examples section ofeach incorporated herein by reference.

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering to a subject a therapeuticallyeffective amount of an antibody-drug conjugate (ADC), such as IMMU-132,as described herein. Preferably the subject has a Trop-2 positive cancerthat is resistant to therapy with checkpoint inhibitor antibodies. TheADCs can be given once or repeatedly, depending on the disease state andtolerability of the conjugate, and can also be used optionally incombination with other therapeutic modalities, such as surgery, externalradiation, radioimmunotherapy, immunotherapy, chemotherapy, antisensetherapy, interference RNA therapy, gene therapy, and the like. Eachcombination will be adapted to the tumor type, stage, patient conditionand prior therapy, and other factors considered by the managingphysician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, therapeutic conjugates comprising ananti-TROP-2 antibody such as the hRS7 MAb can be used to treatcarcinomas such as carcinomas of the esophagus, pancreas, lung, stomach,colon and rectum, urinary bladder, breast, ovary, uterus, kidney andprostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964 and8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3(QQHYITPLT, SEQ ID NO:3) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV,SEQ ID NO:6).

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In a preferred embodiment, a more effective incorporation into cells canbe accomplished by using multivalent, multispecific or multivalent,monospecific antibodies. Examples of such bivalent and bispecificantibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655; 7,238,785;and 7,282,567, the Examples section of each of which is incorporatedherein by reference. These multivalent or multispecific antibodies areparticularly preferred in the targeting of cancers and infectiousorganisms (pathogens), which express multiple antigen targets and evenmultiple epitopes of the same antigen target, but which often evadeantibody targeting and sufficient binding for immunotherapy because ofinsufficient expression or availability of a single antigen target onthe cell or pathogen. By targeting multiple antigens or epitopes, saidantibodies show a higher binding and residence time on the target, thusaffording a higher saturation with the drug being targeted in thisinvention.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelali sib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839. Such agents may be part of theconjugates described herein or may alternatively be administered incombination with the described conjugates, either prior to,simultaneously with or after the conjugate.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -ß; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-ß; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-ß; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

In certain embodiments, a therapeutic agent to be used in combinationwith an anti-Trop-2 ADC is a microtubule inhibitor, such as a vincaalkaloid, a taxanes, a maytansinoid or an auristatin. Exemplary knownmicrotubule inhibitors include paclitaxel, vincristine, vinblastine,mertansine, epothilone, docetaxel, discodermolide, combrestatin,podophyllotoxin, CI-980, phenylahistins, steganacins, curacins,2-methoxy estradiol, E7010, methoxy benzenesuflonamides, vinorelbine,vinflunine, vindesine, dolastatins, spongistatin, rhizoxin, tasidotin,halichondrins, hemiasterlins, cryptophycin 52, MMAE and eribulinmesylate.

In an alternative embodiment, a therapeutic agent to be used incombination with an ADC is a PARP inhibitor, such as olaparib,talazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290,ABT-888, AG014699, BSI-201, CEP-8983 or 3-aminobenzamide.

In another alternative, a therapeutic agent used in combination with anADC is a Bruton tyrosine kinase inhibitor, such as such as ibrutinib(PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 orRN486.

In yet another alternative, a therapeutic agent used in combination withan ADC is a PI3K inhibitor, such as idelalisib, Wortmannin,demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946,BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147,XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115,CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002.

The person of ordinary skill will realize that the subject ADCs,comprising a camptothecin conjugated to an antibody or antibodyfragment, may be used alone or in combination with one or more othertherapeutic agents, such as a second antibody, second antibody fragment,second immunoconjugate, radionuclide, toxin, drug, chemotherapeuticagent, radiation therapy, chemokine, cytokine, immunomodulator, enzyme,hormone, oligonucleotide, RNAi or siRNA. Such additional therapeuticagents may be administered separately, in combination with, or attachedto the subject antibody-drug ADCs.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

ADCs can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the ADC is combined in amixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

In a preferred embodiment, the ADC is formulated in Good's biologicalbuffer (pH 6-7), using a buffer selected from the group consisting ofN-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The ADC can be formulated for intravenous administration via, forexample, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Where subcutaneous administration isdesired, the antibody may be concentrated, for example as disclosed inU.S. Pat. No. 9,180,205, the Examples section of which is incorporatedherein by reference. Subcutaneous injections may be administered as 1,2, or 3 ml injections, which may be administered at a single site or attwo or more sites. Each injection would typically contain between 1.5and 4 mg/kg of concentrated ADC.

Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the ADC. For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of an ADC from sucha matrix depends upon the molecular weight of the ADC, the amount of ADCwithin the matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

Generally, the dosage of an administered ADC for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of ADC that is in therange of from about 1 mg/kg to 24 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy. Preferred dosages may include, but are not limited to, 1 mg/kg,2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg,10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. Any amountin the range of 1 to 24 mg/kg may be used. The dosage is preferablyadministered multiple times, once or twice a week. A minimum dosageschedule of 4 weeks, more preferably 8 weeks, more preferably 16 weeksor longer may be used. The schedule of administration may compriseadministration once or twice a week, on a cycle selected from the groupconsisting of: (i) weekly; (ii) every other week; (iii) one week oftherapy followed by two, three or four weeks off; (iv) two weeks oftherapy followed by one, two, three or four weeks off; (v) three weeksof therapy followed by one, two, three, four or five week off; (vi) fourweeks of therapy followed by one, two, three, four or five week off;(vii) five weeks of therapy followed by one, two, three, four or fiveweek off; and (viii) monthly. The cycle may be repeated 4, 6, 8, 10, 12,16 or 20 times or more.

Alternatively, an ADC may be administered as one dosage every 2 or 3weeks, repeated for a total of at least 3 dosages. Or, twice per weekfor 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m²(340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kgpatient), it may be administered once or even twice weekly for 4 to 10weeks. Alternatively, the dosage schedule may be decreased, namely every2 or 3 weeks for 2-3 months. It has been determined, however, that evenhigher doses, such as 12 mg/kg once weekly or once every 2-3 weeks canbe administered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the ADCs are of use for therapy of cancer.Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenström's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with ADCs may include acute andchronic immune thrombocytopenias, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis, bullouspemphigoid, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one ADC or other targeting moiety as described herein. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1. Production and Use of Anti-Trop-2-SN-38 Antibody-DrugConjugate

The humanized RS7 (hRS7) anti-Trop-2 antibody was produced as describedin U.S. Pat. No. 7,238,785, the Figures and Examples section of whichare incorporated herein by reference. SN-38 attached to a CL2A linkerwas produced and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac),hA20 (anti-CD20) or hMN-14 (anti-CEACAM5) antibodies according to U.S.Pat. No. 7,999,083 (Example 10 and 12 of which are incorporated hereinby reference). The conjugation protocol resulted in a ratio of about 6SN-38 molecules attached per antibody molecule.

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. In a Capan 1 pancreatic tumor model, specific CL2A-SN-38conjugates of hRS7 (anti-Trop-2), hPAM4 (anti-MUC-5ac), and hMN-14(anti-CEACAM5) antibodies showed better efficacies than controlhA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control (not shown).Similarly in a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (not shown).

Example 2. Therapeutic Use of Anti-Trop-2 ADC (Sacituzumab Govitecan) inPatients Refractory to Checkpoint Inhibitor Therapy

Summary

IMMU-132 (sacituzumab govitecan, aka hRS7-CL2A-SN-38) has shownpromising therapeutic results in phase II trials of patients withmetastatic triple-negative breast and other cancers who were heavilypretreated (ClinicalTrials.gov, NCT01631552), and who express highlevels of Trop-2. This novel Trop-2-targeting humanized antibody isconjugated with 7.6 moles of SN-38, the active form of irinotecan, bythe CL2A linker discussed above, and is less glucuronidated in vivo thanirinotecan, accounting for a significantly lower incidence of diarrheain patients treated with the agent.

Surprisingly, IMMU-132 is highly efficacious in patients who havepreviously relapsed from or shown resistance to many standardanti-cancer therapies, including the parent compound irinotecan. A newclass of anti-cancer agents, known as checkpoint inhibitors, includeantibodies or other inhibitory agents against cytotoxic T-lymphocyteantigen 4 (CTLA-4), programmed cell death protein (PD-1) and programmedcell death ligand 1 (PD-L1). As described herein, IMMU-132 showssurprising and unexpected efficacy against tumors that arerelapsed/refractory to checkpoint inhibitors as well as otheranti-cancer agents.

The data below summarizes results for an exemplary case study of apatient (255-029) who responded to therapy with IMMU-132 after havingshown resistance to a PD-L1 inhibitor.

Cancer: TNBC (patient progressed after PD-L1 therapy)

Best Response: Partial response, confirmed (54% shrinkage)

IMMU-132 starting dose: 10 mg/kg

Number IMMU-132 treatments: 40+

Time to Progression: 12.4+ months (not reached)

End of study: Continuing treatment

Medical History—

The patient is a 47-year old female who was first diagnosed in October2007 with a 1.8 cm mass in her right breast. She underwent a rightmastectomy with a sentinel node biopsy (no malignancy in nodes). Thetumor was determined to be negative for ER, PR and Her-2 (i.e., TNBC).She proceeded with 4 cycles of AC (doxorubicin and cyclophosphamide)therapy, followed by weekly low dose paclitaxel (×12). In October 2008,a CT study showed a previously unnoticed pulmonary nodule and anenlarged mammary node; the node was cancer. She had local surgery toremove the node and then received XRT (radiation therapy) through March2009. In June 2010 CT documented recurrent pulmonary and boneinvolvement. The patient enrolled in a clinical trial using AVASTIN®(bevacizumab), METMAB® (onartuzumab, anti-hepatocyte growth factor), andTAXOL® (paclitaxel), which she was maintained on until March 2013,having a complete response, but eventual progression of peripheral lungnodule and node in AP (aortopulmonary) window. In September 2014, shestarted on PD-L1 antibody (MPDL3280A) for 6 cycles, but progressed inJanuary 2015. She was then referred to the IMMU-132 trial.

IMMU-132 Treatment—

The patient initiated treatment on 6 Feb. 2015 at a dose of 10 mg/kg,and has continued to receive this dose without reduction or delay inplanned schedule now for 40+ doses.

Results

The patient initially presented with 3 target lesions (1 lung, 2 lymphnodes in the chest) with a sum of diameters equaling 60 mm, and anadditional non-target lesion (another lymph node in the chest) (FIG. 2).On first assessment (7 Apr. 2015), the patient's sum of the targetlesion diameters decreased 33%, qualifying as a RECIST 1.1 partialresponse. A confirmatory follow-CT performed ˜5 weeks later (18 May2015) showed the response improving, with a 48% overall reduction (FIG.3, FIG. 4). On 19 Jul. 2015, CT showed the patient continued with apartial response, having 50% shrinkage. A CT on 13 Sep. 2015 showed acontinuing PR with a 52% shrinkage, and one on 15 Nov. 15 showed a 54%shrinkage. The latest CT performed on 25 Feb. 2016 showed the PR wascontinuing with a 46% shrinkage.

A summary of CT results is shown in FIG. 1. Baseline CT scans are shownin FIG. 2, with axial images on the top row and sagittal images on thebottom row (tumors indicated by arrows). A direct comparison of thetarget lesions is seen in FIG. 3 and FIG. 4. FIG. 3 shows both targetlesions 1 and 2, taken in the same plane. Baseline (29 Jan. 2015) isshown on top. A significant shrinkage in both L1 and L2 was apparent onthe second response assessment (19 May 2015), shown on the bottom. FIG.4 shows the equivalent images for target lesion 3, with baseline on topand second response assessment on the bottom. The patient is continuingtreatment and continues to show a partial response to IMMU-132, afterpreviously failing checkpoint inhibitor therapy.

Immunohistochemical analysis for Trop-2 expression of the patient'stumors showed positive expression, with a staining level of 2+(data notshown).

Toxicities—

As of the date of this report, the most severe adverse effect has been aG3 hypophosphatemia (not related), with likely related toxicities beinga G2 neutropenia 2 Apr. 15; dose 6), fatigue (G1), nausea (G1), amaculo-popular rash (G1), alopecia (G2), and rhinorrhea (G1).

These results demonstrate that IMMU-132 is highly efficacious inpatients who had previously been relapsed from or resistant tocheckpoint inhibitor therapy. At a therapeutic dosage of IMMU-132, thepatient showed only manageable toxicities. These results show theutility of IMMU-132 for Trop-2 positive cancers, such as triple negativebreast cancer (TNBC).

Example 3. ADCC Activity of Anti-Trop-2 ADCs

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (not shown). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM.

Overall activity was low, but significant (not shown). There was 8.5%specific lysis for the hRS7 IgG which was not significantly differentfrom hRS7-Pro-2-PDox. Both were significantly better than hLL2 controland hRS7-NEM and sacituzumab govitecan (P<0.02, two-tailed t-test).There was no difference between hRS7-NEM and sacituzumab govitecan.

Example 4. Efficacy of Anti-Trop-2-SN-38 ADC Against Diverse EpithelialCancers In Vivo

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 (hRS7) ADC against several human solid tumor types,and to assess its tolerability in mice and monkeys, the latter withtissue cross-reactivity to hRS7 similar to humans. Two SN-38derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The ADCs were characterized invitro for stability, binding, and cytotoxicity. Efficacy was tested infive different human solid tumor-xenograft models that expressed Trop-2antigen. Toxicity was assessed in mice and in Cynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced bysacituzumab govitecan at nontoxic doses in mice bearing Calu-3 (P≤0.05),Capan-1 (P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) whencompared to nontargeting control ADCs. Mice tolerated a dose of 2×12mg/kg (SN-38 equivalents) with only short-lived elevations in ALT andAST liver enzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kgexhibited only transient decreases in blood counts, although,importantly, the values did not fall below normal ranges.

In summary, the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant andspecific antitumor effects against a range of human solid tumor types.It was well tolerated in monkeys, with tissue Trop-2 expression similarto humans, at clinically relevant doses.

Introduction

Successful irinotecan treatment of patients with solid tumors has beenlimited, due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≤3.2 mg/kg cumulative SN-38 equivalent dose). Trop-2 is widelyexpressed in many epithelial cancers, but also some normal tissues, andtherefore a dose escalation study in Cynomolgus monkeys was performed toassess the clinical safety of this conjugate. Monkeys tolerated 24 mgSN-38 equivalents/kg with only minor, reversible, toxicities. Given itstumor-targeting and safety profile, sacituzumab govitecan provides asignificant improvement in the management of solid tumors responsive toirinotecan.

Material and Methods

Cell Lines, Antibodies, and Chemotherapeutics—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection. These include Calu-3 (non-small celllung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205(colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreaticadenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22(hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc.Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

SN-38 ADCs and In Vitro Aspects—

Synthesis of CL2-SN-38 has been described previously (Moon et al., 2008,J Med Chem 51:6916-26). Its conjugation to hRS7 IgG and serum stabilitywere performed as described (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Chem Res 15:6052-61). Preparations ofCL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding,and cytotoxicity studies, were conducted as described in the precedingExamples.

In Vivo Therapeutic Studies—

For all animal studies, the doses of SN-38 ADCs and irinotecan are shownin SN-38 equivalents. Based on a mean SN-38/IgG substitution ratio of 6,a dose of 500 μg ADC to a 20-g mouse (25 mg/kg) contains 0.4 mg/kg ofSN-38. Irinotecan doses are likewise shown as SN-38 equivalents (i.e.,40 mg irinotecan/kg is equivalent to 24 mg/kg of SN-38).

NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, and maleSwiss-Webster mice, 10 weeks old, were purchased from Taconic Farms.Tolerability studies were performed in Cynomolgus monkeys (Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd. Animals wereimplanted subcutaneously with different human cancer cell lines. Tumorvolume (TV) was determined by measurements in 2 dimensions usingcalipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized sacituzumab govitecan and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics—

Growth curves are shown as percent change in initial TV over time.Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P≤0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and Biodistribution—

¹¹¹In-radiolabeled sacituzumab govitecan and hRS7 IgG were injected intonude mice bearing s.c. SK-MES-1 tumors (˜0.3 cm³). One group wasinjected intravenously with 20 μCi (250-μg protein) of ¹¹¹In-sacituzumabgovitecan, whereas another group received 20 μCi (250-μg protein) of¹¹¹In-hRS7 IgG. At various timepoints mice (5 per timepoint) wereanesthetized, bled via intracardiac puncture, and then euthanized.Tumors and various tissues were removed, weighed, and counted by γscintillation to determine the percentage injected dose per gram tissue(% ID/g). A third group was injected with 250 μg of unlabeledsacituzumab govitecan 3 days before the administration of¹¹¹In-sacituzumab govitecan and likewise necropsied. A 2-tailed t-testwas used to compare sacituzumab govitecan and hRS7 IgG uptake afterdetermining equality of variance using the f-test. Pharmacokineticanalysis on blood clearance was performed using WinNonLin software(Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys—

Briefly, mice were sorted into 4 groups each to receive 2-mL i.p.injections of either a sodium acetate buffer control or 3 differentdoses of sacituzumab govitecan (4, 8, or 12 mg/kg of SN-38) on days 0and 3 followed by blood and serum collection, as described in Results.Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2different doses of sacituzumab govitecan. Dosages, times, and number ofmonkeys bled for evaluation of possible hematologic toxicities and serumchemistries are described in the Results.

Results

Stability and Potency of hRS7-SN-38—

Two different linkages were used to conjugate SN-38 to hRS7 IgG (notshown). The first is termed CL2-SN-38 and has been described previously(Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, ClinChem Res 15:6052-61). A change in the synthesis of CL2 to remove thephenylalanine moiety within the linker was used to produce the CL2Alinker. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity. This result was surprising, since the phenylalanineresidue in CL2 is part of a designed cleavage site for cathepsin B, alysosomal protease.

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, sacituzumab govitecan and hRS7-CL2-SN-38 werecompared in mice bearing COLO 205 (not shown) or Capan-1 tumors (notshown), using 0.4 mg or 0.2 mg/kg SN-38 twice weekly×4 weeks,respectively, and with starting tumors of 0.25 cm³ size in both studies.Both the sacituzumab govitecan and CL2-SN-38 conjugates significantlyinhibited tumor growth compared to untreated (AUC_(14days) P<0.002 vs.saline in COLO 205 model; AUC_(21days) P<0.001 vs. saline in Capan-1model), and a nontargeting anti-CD20 control ADC, hA20-CL2A-SN-38(AUC_(14days) P<0.003 in COLO-205 model; AUC_(35days): P<0.002 inCapan-1 model). At the end of the study (day 140) in the Capan-1 model,50% of the mice treated with sacituzumab govitecan and 40% of thehRS7-CL2-SN-38 mice were tumor-free, whereas only 20% of thehA20-ADC-treated animals had no visible sign of disease. The CL2A linkerresulted in a higher efficacy compared to CL2 (not shown).

Mechanism of Action—

In vitro cytotoxicity studies demonstrated that sacituzumab govitecanhad IC₅₀ values in the nmol/L range against several different solidtumor lines (Table 2). The IC₅₀ with free SN-38 was lower than theconjugate in all cell lines. Although there was no apparent correlationbetween Trop-2 expression and sensitivity to sacituzumab govitecan, theIC50 ratio of the ADC versus free SN-38 was lower in the higherTrop-2-expressing cells, most likely reflecting the enhanced ability tointernalize the drug when more antigen is present.

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis (e.g., Cusack et al., 2001, Cancer Res 61:3535-40; Liu etal. 2009, Cancer Lett 274:47-53; Lagadec et al., 2008, Br J Cancer98:335-44). Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression (not shown), whereas sacituzumab govitecanresulted in only a 10-fold increase (not shown), a finding consistentwith the higher activity with free SN-38 in this cell line (Table 2).However, sacituzumab govitecan increased p21^(Waf1/Cip1) expression inCalu-3 more than 2-fold over free SN-38 (not shown).

A greater disparity between sacituzumab govitecan- and freeSN-38-mediated signaling events was observed in p53 expression (notshown). In both BxPC-3 and Calu-3, upregulation of p53 with free SN-38was not evident until 48 hours, whereas sacituzumab govitecanupregulated p53 within 24 hours (not shown). In addition, p53 expressionin cells exposed to the ADC was higher in both cell lines compared toSN-38 (not shown). Interestingly, although hRS7 IgG had no appreciableeffect on p21^(Waf1/Cip1) expression, it did induce the upregulation ofp53 in both BxPC-3 and Calu-3, but only after a 48-hour exposure (notshown). In terms of later apoptotic events, cleavage of PARP was evidentin both cell lines when incubated with either SN-38 or the conjugate(not shown). The presence of the cleaved PARP was higher at 24 hours inBxPC-3 (not shown), which correlates with high expression of p21 and itslower IC₅₀. The higher degree of cleavage with free SN-38 over the ADCwas consistent with the cytotoxicity findings.

Efficacy of hRS7-SN-38—

Because Trop-2 is widely expressed in several human carcinomas, studieswere performed in several different human cancer models, which startedusing the hRS7-CL2-SN-38 linkage, but later, conjugates with theCL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mg SN-38/kgof the hRS7-CL2-SN-38 every 4 days×4 had a significantly improvedresponse compared to animals administered the equivalent amount ofnon-targeting hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs. 0.80±0.91 cm³,respectively; AUC_(42days) P<0.026; not shown). A dose-response wasobserved when the dose was increased to 0.4 mg/kg SN-38 (not shown). Atthis higher dose level, all mice given the specific hRS7 conjugate were“cured” within 28 days, and remained tumor-free until the end of thestudy on day 147, whereas tumors regrew in animals treated with theirrelevant ADC (specific vs. irrelevant AUC_(98days): P=0.05). In micereceiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5-foldby day 56 (TV=1.10±0.88 cm³; AUC_(56days) P<0.006 vs. hRS7-CL2-SN-38)(not shown).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (notshown), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth overthe 28-day treatment period with significantly smaller tumors comparedto control anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09cm³, 1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28days)P<0.016).

TABLE 2 Expression of Trop-2 in vitro cytotoxicity of SN-38 andhRS7-SN-38 in various solid tumor lines Trop-2 expression via FACSCytotoxicity results Median SN-38 95% CI hRS7-SN-38 95% CI ADC/freefluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ SN-38 Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) ratio Calu-3 282.2 (4.7)99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95 1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99 5.02-9.72 2.00 PC-3 46.2 (5.5) 73.6% 1.86 1.16-2.99 4.242.99-6.01 2.28 SK-MES-1 44.0 (3.5) 91.2% 8.61  6.30-11.76 23.1417.98-29.78 2.69 BxPC-3 26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.982.80

The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38 in COLO 205 cells, because mouse serum can moreefficiently convert irinotecan to SN-38 (Morton et al., 2000, Cancer Res60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 μgcumulative) was 37.5-fold greater than with the conjugate (64 μg total).

Animals bearing Capan-1 (not shown) showed no significant response toirinotecan alone when given at an SN-38-dose equivalent to thehRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was0.04±0.05 cm³ in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62cm³ in irinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001; not shown). When the irinotecan dose was increased 10-fold to 4mg/kg SN-38, the response improved, but still was not as significant asthe conjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49) P<0.001) (not shown). An equal dose ofnontargeting hA20-CL2-SN-38 also had a significant antitumor effect ascompared to irinotecan-treated animals, but the specific hRS7 conjugatewas significantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49)P<0.018) (not shown).

Studies with the sacituzumab govitecan ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (not shown), sacituzumab govitecan again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively; AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38 equivalent dose(TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively; AUC_(day25) P<0.004)(not shown). Interestingly, in mice bearing SK-MES-1 human squamous celllung tumors treated with 0.4 mg/kg of the ADC (not shown), tumor growthinhibition was superior to saline or unconjugated hRS7 IgG (TV=0.36±0.25cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively; AUC_(28days),P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD of irinotecanprovided the same antitumor effects as the specific hRS7-SN-38 conjugate(not shown). In all murine studies, the hRS7-SN-38 ADC was welltolerated in terms of body weight loss (not shown).

Biodistribution of Sacituzumab Govitecan—

The biodistributions of sacituzumab govitecan or unconjugated hRS7 IgGwere compared in mice bearing SK-MES-1 human squamous cell lungcarcinoma xenografts (not shown), using the respective ¹¹¹In-labeledsubstrates. A pharmacokinetic analysis was performed to determine theclearance of sacituzumab govitecan relative to unconjugated hRS7 (notshown). The ADC cleared faster than the equivalent amount ofunconjugated hRS7, with the ADC exhibiting ˜40% shorter half-life andmean residence time. Nonetheless, this had a minimal impact on tumoruptake (not shown). Although there were significant differences at the24- and 48-hour timepoints, by 72 hours (peak uptake) the amounts ofboth agents in the tumor were similar. Among the normal tissues, hepaticand splenic differences were the most striking (not shown). At 24 hourspostinjection, there was >2-fold more sacituzumab govitecan in the liverthan hRS7 IgG (not shown). Conversely, in the spleen there was 3-foldmore parental hRS7 IgG present at peak uptake (48-hour timepoint) thansacituzumab govitecan (not shown). Uptake and clearance in the rest ofthe tissues generally reflected differences in the blood concentration(not shown).

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of ¹¹¹In-sacituzumabgovitecan in predosed mice was substantially reduced at every timepointin comparison to animals that did not receive the predose (e.g., at 72hours, predosed tumor uptake was 12.5%±3.8% ID/g vs. 25.4%±8.1% ID/g inanimals not given the predose; P=0.0123; not shown). Predosing had noappreciable impact on blood clearance or tissue uptake (not shown).These studies suggest that in some tumor models, tumor accretion of thespecific antibody can be reduced by the preceding dose(s), which likelyexplains why the specificity of a therapeutic response could bediminished with increasing ADC doses and why further dose escalation isnot indicated.

Tolerability of Sacituzumab Govitecan in Swiss-Webster Mice andCynomolgus Monkeys

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of sacituzumab govitecan, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transaminase (AST, not shown) andalanine transaminase (ALT, not shown). Seven days after treatment, ASTrose above normal levels (>298 U/L) in all 3 treatment groups (notshown), with the largest proportion of mice being in the 2×8 mg/kggroup. However, by 15 days posttreatment, most animals were within thenormal range. ALT levels were also above the normal range (>77 U/L)within 7 days of treatment (not shown) and with evidence ofnormalization by Day 15. Livers from all these mice did not showhistologic evidence of tissue damage (not shown). In terms of renalfunction, only glucose and chloride levels were somewhat elevated in thetreated groups. At 2×8 mg/kg, 5 of 7 mice had slightly elevated glucoselevels (range of 273-320 mg/dL, upper end of normal 263 mg/dL) thatreturned to normal by 15 days postinjection. Similarly, chloride levelswere slightly elevated, ranging from 116 to 127 mmol/L (upper end ofnormal range 115 mmol/L) in the 2 highest dosage groups (57% in the 2×8mg/kg group and 100% of the mice in the 2×12 mg/kg group), and remainedelevated out to 15 days postinjection. This also could be indicative ofgastrointestinal toxicity, because most chloride is obtained throughabsorption by the gut; however, at termination, there was no histologicevidence of tissue damage in any organ system examined (not shown).

Because mice do not express the Trop-2 target antigen of hRS7 in normaltissues, a more suitable model was required to determine the potentialof the hRS7 conjugate for clinical use. Immunohistology studies revealedbinding in multiple tissues in both humans and Cynomolgus monkeys(breast, eye, gastrointestinal tract, kidney, lung, ovary, fallopiantube, pancreas, parathyroid, prostate, salivary gland, skin, thymus,thyroid, tonsil, ureter, urinary bladder, and uterus; not shown). Basedon this cross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of sacituzumab govitecan had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (not shown), but values did not fall belownormal ranges. No abnormal values were found in the serum chemistries.Histopathology of the animals necropsied on day 11 (8 days after lastinjection) showed microscopic changes in hematopoietic organs (thymus,mandibular and mesenteric lymph nodes, spleen, and bone marrow),gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum,colon, and rectum), female reproductive organs (ovary, uterus, andvagina), and at the injection site. These changes ranged from minimal tomoderate and were fully reversed at the end of the recovery period (day32) in all tissues, except in the thymus and gastrointestinal tract,which were trending towards full recovery at this later timepoint (notshown).

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group (not shown). These dataindicate that dose-limiting toxicities were identical to that ofirinotecan; namely, intestinal and hematologic. Thus, the MTD forsacituzumab govitecan lies between 2×0.96 and 1.92 mg SN-38/kg, whichrepresents a human equivalent dose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents (Ohmachi etal., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer99:1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14). TheRS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995,Cancer Res 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38(Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. To simplify the synthetic process, inCL2A the phenylalanine was eliminated, and thus the cathepsin B cleavagesite was removed. Interestingly, this product had a better-definedchromatographic profile compared to the broad profile obtained with CL2(not shown), but more importantly, this change had no impact on theconjugate's binding or stability, and surprisingly produced a smallincrease in potency in side-by-side testing.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96, Zhao etal., 2008, Bioconjug Chem 19:849-59) and NK012 (Koizumi et al., 2006,Cancer Res 66:10048-56). ENZ-2208 utilizes a branched PEG to link about3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micellenanoparticle containing 20% SN-38 by weight. With our ADC, thisdisparity (i.e., ratio of potency with free vs. conjugated SN-38)decreased as the Trop-2 expression levels increased in the tumor cells,suggesting an advantage to targeted delivery of the drug. In terms of invitro serum stability, both the CL2- and CL2A-SN-38 forms of hRS7-SN-38yielded a t/_(1/2) of ˜20 hours, which is in contrast to the shortt/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56). Treatment of tumor-bearing mice withhRS7-SN-38 (either with CL2-SN-38 or CL2A-SN-38) significantly inhibitedtumor growth in 5 different tumor models. In 4 of them, tumorregressions were observed, and in the case of Calu-3, all mice receivingthe highest dose of hRS7-SN-38 were tumor-free at the conclusion ofstudy. Unlike in humans, irinotecan is very efficiently converted toSN-38 by a plasma esterase in mice, with a greater than 50% conversionrate, and yielding higher efficacy in mice than in humans (Morton etal., 2000, Cancer Res 60:4206-10; Furman et al., 1999, J Clin Oncol17:1815-24). When irinotecan was administered at 10-fold higher orequivalent SN-38 levels, hRS7-SN-38 was significantly better incontrolling tumor growth. Only when irinotecan was administered at itsMTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal theeffectiveness of hRS7-SN-38. In patients, we would expect this advantageto favor sacituzumab govitecan even more, because the bioconversion ofirinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues (Jain, 1994, Sci Am 271:58-61). With our conjugate, 50%of the SN-38 will be released in ˜13 hours when the pH is lowered to alevel mimicking lysosomal levels (e.g., pH 5.3 at 37° C.; data notshown), whereas at the neutral pH of serum, the release rate is reducednearly 2-fold. If an irrelevant conjugate enters an acidic tumormicroenvironment, it is expected to release some SN-38 locally. Otherfactors, such as tumor physiology and innate sensitivities to the drug,will also play a role in defining this “baseline” activity. However, aspecific conjugate with a longer residence time should have enhancedpotency over this baseline response as long as there is ample antigen tocapture the specific antibody. Biodistribution studies in the SK-MES-1model also showed that if tumor antigen becomes saturated as aconsequence of successive dosing, tumor uptake of the specific conjugateis reduced, which yields therapeutic results similar to that found withan irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5 (Sapra et al., 2008, Clin Cancer Res 14:1888-96; Pastorini etal., 2010, Clin Cancer Res 16:4809-21), and preclinical studies withNK012 involved its MTD of 30 mg/kg×3 (Koizumi et al., 2006, Cancer Res66:10048-56). Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

Biodistribution studies revealed that sacituzumab govitecan had similartumor uptake as the parental hRS7 IgG, but cleared substantially fasterwith 2-fold higher hepatic uptake, which may be due to thehydrophobicity of SN-38. With the ADC being cleared through the liver,hepatic and gastrointestinal toxicities were expected to be doselimiting. Although mice had evidence of increased hepatic transaminases,gastrointestinal toxicity was mild at best, with only transient loss inweight and no abnormalities noted upon histopathologic examination.Interestingly, no hematological toxicity was noted. However, monkeysshowed an identical toxicity profile as expected for irinotecan, withgastrointestinal and hematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wasimportant to perform toxicity studies in monkeys that have a similartissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose(˜12 mg/m2) with mild and reversible toxicity, which extrapolates to ahuman dose of ˜0.3 mg/kg/dose (˜11 mg/m2). In a Phase I clinical trialof NK012, patients with solid tumors tolerated 28 mg/m2 of SN-38 every 3weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchiet al., 2010, Clin Cancer Res 16:5058-66). Similarly, Phase I clinicaltrials with ENZ-2208 revealed dose-limiting febrile neutropenia, with arecommendation to administer 10 mg/m² every 3 weeks or 16 mg/m² ifpatients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTCInternational Conference on Molecular Targets and Cancer Therapeutics:2009 Nov. 15-19: Boston, Mass.; Poster No C216: Patnaik et al.,AACR-NCI-EORTC International Conference on Molecular Targets and CancerTherapeutics; 2009 Nov. 15-19; Boston, Mass.; Poster No C221). Becausemonkeys tolerated a cumulative human equivalent dose of 22 mg/m2, itappears that even though hRS7 binds to a number of normal tissues, theMTD for a single treatment of the hRS7 ADC could be similar to that ofthe other nontargeting SN-38 agents. Indeed, the specificity of theanti-Trop-2 antibody did not appear to play a role in defining the DLT,because the toxicity profile was similar to that of irinotecan. Moreimportantly, if antitumor activity can be achieved in humans as in micethat responded with human equivalent dose of just at 0.03 mg SN-38equivalents/kg/dose, then significant antitumor responses may berealized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 5. Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained for ADC conjugation. The first, 162-46.2, was purified from ahybridoma (ATCC, HB-187) grown up in roller-bottles. A second antibody,MAB650, was purchased from R&D Systems (Minneapolis, Minn.). For acomparison of binding, the Trop-2 positive human gastric carcinoma,NCI-N87, was used as the target. Cells (1.5×10⁵/well) were plated into96-well plates the day before the binding assay. The following morning,a dose/response curve was generated with 162-46.2, MAB650, and murineRS7 (0.03 to 66 nM). These primary antibodies were incubated with thecells for 1.5 h at 4° C. Wells were washed and an anti-mouse-HRPsecondary antibody was added to all the wells for 1 h at 4° C. Wells arewashed again followed by the addition of a luminescence substrate.Plates were read using Envision plate reader and values are reported asrelative luminescent units.

All three antibodies had similar K_(D)-values of 0.57 nM for RS7, 0.52nM for 162-46.2 and 0.49 nM for MAB650. However, when comparing themaximum binding (B_(max)) of 162-46.2 and MAB650 to RS7 they werereduced by 25% and 50%, respectively (B_(Max) 11,250 for RS7, 8,471 for162-46.2 and 6,018 for MAB650) indicating different binding propertiesin comparison to RS7.

Example 6. Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and sacituzumab govitecan ADCsusing two different human pancreatic adenocarcinoma cell lines (BxPC-3and Capan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto sacituzumab govitecan, MAB650-SN-38, and free SN-38 at a drug rangeof 3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a controlat protein equivalent doses as the MAB650-SN-38. Plates were incubatedat 37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

Sacituzumab govitecan and MAB650-SN-38 had similar growth-inhibitoryeffects with IC50-values in the low nM range which is typical forSN-38-ADCs in these cell lines (not shown). In the human Capan-1pancreatic adenocarcinoma cell line (not shown), the sacituzumabgovitecan ADC showed an IC₅₀ of 3.5 nM, compared to 4.1 nM for theMAB650-SN-38 ADC and 1.0 nM for free SN-38. In the human BxPC-3pancreatic adenocarcinoma cell line (not shown), the sacituzumabgovitecan ADC showed an IC₅₀ of 2.6 nM, compared to 3.0 nM for theMAB650-SN-38 ADC and 1.0 nM for free SN-38. In the human NCI-N87 gastricadenocarcinoma cell line (not shown), the sacituzumab govitecan ADCshowed an IC₅₀ of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADCand 4.3 nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for sacituzumab govitecan.

Example 7. Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to sacituzumab govitecan, 162-46.2-SN-38, or freeSN-38 at a drug range of 3.84×10⁻¹² to 2.5×10⁻⁷M. Unconjugated 162-46.2and hRS7 were used as controls at the same protein equivalent doses asthe 162-46.2-SN-38 and sacituzumab govitecan, respectively. Plates wereincubated at 37° C. for 96 h. After this incubation period, an MTSsubstrate was added to all of the plates and read for color developmentat half-hour intervals until untreated control wells had an OD_(492nm)reading of approximately 1.0. Growth inhibition was measured as apercent of growth relative to untreated cells using Microsoft Excel andPrism software (non-linear regression to generate sigmoidal doseresponse curves which yield IC₅₀-values).

The 162-46.2-SN-38 ADC had a similar IC₅₀-values when compared tosacituzumab govitecan (not shown). When tested against the BxPC-3 humanpancreatic adenocarcinoma cell line (not shown), sacituzumab govitecanhad an IC₅₀ of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nMfor free SN-38. When tested against the MDA-MB-468 human breastadenocarcinoma cell line (not shown), sacituzumab govitecan had an IC₅₀of 3.9 nM, compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for freeSN-38. The free antibodies alone showed little cytotoxicity to eitherTrop-2 positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 8. Clinical Trials With IMMU-132 (Sacituzumab Govitecan)Anti-Trop-2 ADC Comprising hRS7 Antibody Conjugated to SN-38

Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(˜1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients whohad failed multiple prior therapies (some including topoisomerase-I/IIinhibiting drugs), and the ongoing Phase II extension now reporting on69 patients, including in colorectal (CRC), small-cell and non-smallcell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC),pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≥2⁺ immunohistochemical staining) in most archivedtumors. In a 3+3 trial design, IMMU-132 was given on days 1 and 8 inrepeated 21-day cycles, starting at 8 mg/kg/dose, then 12 and 18 mg/kgbefore dose-limiting neutropenia. To optimize cumulative treatment withminimal delays, phase II is focusing on 8 and 10 mg/kg (n=30 and 14,respectively). In 49 patients reporting related AE at this time,neutropenia ≥Grade 3 occurred in 28% (4% Grade 4). Most commonnon-hematological toxicities initially in these patients have beenfatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea (47%; ≥G3=9%),alopecia (40%), and vomiting (32%; ≥G3=2%); alopecia also occurredfrequently. Homozygous UGT1A1*28/*28 was found in 6 patients, 2 of whomhad more severe hematological and GI toxicities.

In the Phase I and the expansion phases, there are now 48 patients(excluding PDC) who are assessable by RECIST/CT for best response. Seven(15%) of the patients had a partial response (PR), including patientswith CRC (N=1), TNBC (N=2), SCLC (N=2), NSCLC (N=1), and esophagealcancers (N=1), and another 27 patients (56%) had stable disease (SD),for a total of 38 patients (79%) with disease response; 8 of 13CT-assessable PDC patients (62%) had SD, with a median time toprogression (TTP) of 12.7 wks compared to 8.0 weeks in their last priortherapy. The TTP for the remaining 48 patients is 12.6+ wks (range 6.0to 51.4 wks). Plasma CEA and CA19-9 correlated with responses who hadelevated titers of these antigens in their blood. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months.

The conjugate cleared from the serum within 3 days, consistent with invivo animal studies where 50% of the SN-38 was released daily, with >95%of the SN-38 in the serum being bound to the IgG in a non-glucuronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show thatsacituzumab govitecan is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient pool is provided in Table3.

TABLE 3 Clinical Trial Parameters Dosing Once weekly for 2 weeksadministered every 21 days for up to 8 regimen cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥Grade 2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≥Grade 3 toxicity. Dose level 8, 12, 18mg/kg; later reduced to an intermediate dose level of 10 cohorts mg/kg.Cohort size Standard Phase I [3 + 3] design; expansion includes ~15patients in select cancers. DLT Grade 4 ANC ≥7 d; ≥Grade 3 febrileneutropenia of any duration; G4 Plt ≥5 d; G4 Hgb; Grade 4 N/V/D anyduration/G3 N/V/D for >48 h; G3 infusion-related reactions; related ≥G3non-hematological toxicity. Maximum Maximum dose where ≥2/6 patientstolerate 1^(st) 21-d cycle w/o delay or Acceptable reduction or ≥G3toxicity. Dose (MAD) Patients Metastatic colorectal, pancreas, gastric,esophageal, lung (NSCLC, SCLC), triple-negative breast (TNBC), prostate,ovarian, renal, urinary bladder, head/neck, hepatocellular.Refractory/relapsed after standard treatment regimens for metastaticcancer. Prior irinotecan-containing therapy NOT required for enrollment.No bulky lesion >5 cm. Must be 4 weeks beyond any major surgery, and 2weeks beyond radiation or chemotherapy regimen. Gilbert's disease orknown CNS metastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (not shown) and Time to Progression (TTP; notshown) were determined. To summarize the Best Response data, of 8assessable patients with TNBC (triple-negative breast cancer), therewere 2 PR (partial response), 4 SD (stable disease) and 2 PD(progressive disease) for a total response [PR+SD] of 6/8 (75%). ForSCLC (small cell lung cancer), of 4 assessable patients there were 2 PR,0 SD and 2 PD for a total response of 2/4 (50%). For CRC (colorectalcancer), of 18 assessable patients there were 1 PR, 11 SD and 6 PD for atotal response of 12/18 (67%). For esophageal cancer, of 4 assessablepatients there were 1 PR, 2 SD and 1 PD for a total response of 3/4(75%). For NSCLC (non-small cell lung cancer), of 5 assessable patientsthere were 1 PR, 3 SD and 1 PD for a total response of 4/5 (80%). Overall patients treated, of 48 assessable patients there were 7 PR, 27 SDand 14 PD for a total response of 34/48 (71%). These results demonstratethat the anti-TROP-2 ADC (hRS7-SN-38) showed significant clinicalefficacy against a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 4. As apparent from the data of Table 4, the therapeutic efficacyof sacituzumab govitecan was achieved at dosages of ADC showing anacceptably low level of adverse side effects.

TABLE 4 Related Adverse Events Listing for IMMU-132-01 Criteria: Total≥10% or ≥Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4(9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2(4%) Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0Dysgeusia 15% 0 0 Pyrexia 13% 0 0 Abdominal pain 11% 0 0 Hypokalemia 11%1 (2%) 0 WBC Decrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%)Deep vein thrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

The study reported in Table 4 has continued, with 261 patients enrolledto date. The results (not shown) have generally followed along the linesindicated in Table 4, with only neutropenia showing an incidence ofGrade 3 or higher adverse events of over 10% of the patients tested. Forall other adverse events, the incidence of Grade 3 or higher responseswas less than 10%. This distinguishes the instant ADCs from the greatmajority of ADCs and in certain embodiments, the claimed methods andcompositions relate to anti-Trop-2 ADCs that show efficacy in diversesolid tumors, with an incidence of Grade 3 or higher adverse events ofless than 10% of patients for all adverse events other than neutropenia.In a follow-up study, in a total of 421 samples from 121 patients withbaseline and at least one follow-up sample available, no anti-hRS7 oranti-SN-38 antibody response has been detected, despite repeated cyclesof treatment.

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo SFU. She presented with multiple lesionsprimarily in the liver (3+ Trop-2 by immunohistology), entering thesacituzumab govitecan trial at a starting dose of 8 mg/kg about 1 yearafter initial diagnosis. On her first CT assessment, a PR was achieved,with a 37% reduction in target lesions (not shown). The patientcontinued treatment, achieving a maximum reduction of 65% decrease after10 months of treatment (not shown) with decrease in CEA from 781 ng/mLto 26.5 ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topoisomerase-II inhibitor) that ended after 2 months with no response,followed with topotecan (Topoisomerase-I inhibitor) that ended after 2months, also with no response, she received local XRT (3000 cGy) thatended 1 month later. However, by the following month progression hadcontinued. The patient started with IMMU-132 the next month (12 mg/kg;reduced to 6.8 mg/kg; Trop-2 expression 3+), and after two months ofIMMU-132, a 38% reduction in target lesions, including a substantialreduction in the main lung lesion occurred (not shown). The patientprogressed 3 months later after receiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies. In conclusion, at thedosages used, the primary toxicity was a manageable neutropenia, withfew Grade 3 toxicities. IMMU-132 showed evidence of activity (PR anddurable SD) in relapsed/refractory patients with triple-negative breastcancer, small cell lung cancer, non-small cell lung cancer, colorectalcancer and esophageal cancer, including patients with a previous historyof relapsing on topoisomerase-I inhibitor therapy. These results showefficacy of the anti-Trop-2 ADC in a wide range of cancers that areresistant to existing therapies.

Example 9. Comparative Efficacy of Different Anti-Trop-2 ADCs

The therapeutic efficacy of a murine anti-Trop-2 monoclonal antibody(162-46.2) conjugated with SN-38 was compared to sacituzumab govitecanantibody-drug conjugate (ADC) in mice bearing human gastric carcinomaxenografts (NCI-N87). NCI-N87 cells were expanded in tissue culture andharvested with trypsin/EDTA. Female athymic nude mice were injected s.c.with 200 μL of NCI-N87 cell suspension mixed 1:1 with matrigel such that1×10⁷ cells was administered to each mouse. Once tumors reachedapproximately 0.25 cm³ in size (6 days later), the animals were dividedup into seven different treatment groups of nine mice each. For theSN-38 ADCs, mice received 500 μg i.v. injections once a week for twoweeks. Control mice received the non-tumor targeting hA20-SN-38 ADC atthe same dose/schedule. A final group of mice received only saline andserved as the untreated control. Tumors were measured and mice weighedtwice a week. Mice were euthanized for disease progression if theirtumor volumes exceeded 1.0 cm³ in size.

Mean tumor volumes were determined for the SN-38-ADC treated mice (notshown). As determined by area under the curve (AUC), both sacituzumabgovitecan and 162-46.2-SN-38 significantly inhibited tumor growth whencompared to saline and hA20-SN-38 control mice (P<0.001). Treatment withsacituzumab govitecan achieved stable disease in 7 of 9 mice with meantime to tumor progression (TTP) of 18.4±3.3 days. Mice treated with162-46.2-SN-38 achieved a positive response in 6 of 9 mice with theremaining 3 achieving stable disease. Mean TTP was 24.2±6.0 days, whichis significantly longer than sacituzumab govitecan treated animals(P=0.0382).

These results confirm the in vivo efficacy of different anti-Trop-2 ADCsfor treatment of human gastric carcinoma.

Example 10. Treatment of Patients with Advanced, Metastatic PancreaticCancer with Anti-Trop-2 ADC

Summary

IMMU-132 (sacituzumab govitecan) is an anti-Trop-2 ADC comprising thecancer cell internalizing, humanized, anti-Trop-2 hRS7 antibody,conjugated by a pH-sensitive linker to SN-38, the active metabolite ofirinotecan, at a mean drug-antibody ratio of 7.6. Trop-2 is a type-Itransmembrane, calcium-transducing protein expressed at high density,frequency, and specificity in many epithelial cancers, includingpancreatic ductal adenocarcinoma, with limited normal tissue expression.All 29 pancreatic tumor microarray specimens tested were Trop-2-positiveby immunohistochemistry, and human pancreatic cancer cell lines werefound to express 115 k-891 k Trop-2 copies on the cell membrane.

We reported above the results from the IMMU-132 Phase I study enrollingpatients with 13 different tumor types using a 3+3 design. The Phase Idose-limiting toxicity was neutropenia. Over 80% of 24 assessablepatients in this study had long-term stable disease, with partialresponses (RECIST) observed in patients with colorectal (CRC),triple-negative breast (TNBC), small-cell and non-small cell lung (SCLC,NSCLC), and esophageal (EAC) cancers. The present Example reports theresults from the IMMU-132 Phase I/II study cohort of patients withmetastatic PDC. Patients with PDC who failed a median of 2 priortherapies (range 1-5) were given IMMU-132 on days 1 and 8 in repeated21-day cycles.

In the subgroup of PDC patients (N=15), 14 received priorgemcitabine-containing regimens. Initial toxicity data from 9 patientsfound neutropenia [3 of 9≥G3, 33%; and 1 case of G4 febrileneutropenia), which resulted in dose delays or dose reductions. Twopatients had Grade 3 diarrhea; no patient had Grade 3-4 nausea orvomiting. Alopecia (Grades 1-2) occurred in 5 of 9 patients. Bestresponse was assessable in 13 of 14 patients, with 8 stable disease for8 to 21.4 wks (median 12.7 wks; 11.9 wks all 14 patients). One patientwho is continuing treatment has not yet had their first CT assessment.Five had progressive disease by RECIST; 1 withdrew after just 1 dose dueto clinical progression and was not assessable. Serum CA19-9 titersdecreased in 3 of the patients with stable disease by 23 to 72%. Despitemultiple administrations, none of the patients developed an antibodyresponse to IMMU-132 or SN-38. Peak and trough serum samples showed thatIMMU-132 cleared more quickly than the IgG, which is expected based onthe known local release of SN-38 within the tumor cell. Concentrationsof SN-38-bound to IgG in peak samples from one patient given 12 mg/kg ofIMMU-132 showed levels of ˜4000 ng/mL, which is 40-times higher than theSN-38 titers reported in patients given irinotecan therapy.

We conclude that IMMU-132 is active (long-term stable disease) in 62%(8/13) of PDC patients who failed multiple prior therapies, withmanageable neutropenia and little GI toxicity. Advanced PDC patients canbe given repeated treatment cycles (>6) of 8-10 mg/kg IMMU-132 on days 1and 8 of a 21-day cycle, with some dose adjustments or growth factorsupport for neutropenia in subsequent treatment cycles. These resultsagree with the findings in patients with advanced CRC, TNBC, SCLC,NSCLC, EAC who have shown partial responses and long-term stable diseasewith IMMU-132 administration. In summary, monotherapy IMMU-132 is anovel, efficacious treatment regimen for patients with PDC, includingthose with tumors that were previously resistant to other therapeuticregimens for PDC.

Methods and Results

Trop-2 Expression—

The expression of Trop-2 on the surface of various cancer cell lines wasdetermined by flow cytometry using QUANTBRITE® PE beads. The results fornumber of Trop-2 molecules detected in the different cell lines was:BxPC-3 pancreatic cancer (891,000); NCI-N87 gastric cancer (383,000);MDA-MB-468 breast cancer (341,000); SK-MES-1 squamous cell lung cancer(27,000); Capan-1 pancreatic cancer (115,000); AGS gastric cancer(78,000) COLO 205 colon cancer (52,000). Trop-2 expression was alsoobserved in 29 of 29 (100%) tissue microarrays of pancreaticadenocarcinoma (not shown).

SN-38 Accumulation—

SN-38 accumulation was determined in nude mice bearing Capan-1 humanpancreatic cancer xenografts (˜0.06-0.27 g). Mice were injected IV withirinotecan 40 mg/kg (773 μg; Total SN-38 equivalents=448 μg). This doseis MTD in mice. Human dose equivalent=3.25 mg/kg or ˜126 mg/m². Or micewere injected IV with IMMU-132 1.0 mg (SN-38:antibody ratio=7.6; SN-38equivalents=20 μg). This dose is well below the MTD in mice. Humanequivalent dose ˜4 mg/kg IMMU-132 (˜80 μg/kg SN-38 equivalents).Necropsies were performed on 3 animals per interval, in irinotecaninjected mice at 5 min, 1, 2, 6 and 24 hours or in IMMU-132 injectedmice at 1, 6, 24, 48 and 72 h. Tissues were extracted and analyzed byreversed-phase HPLC analysis for SN-38, SN-38G, and irinotecan. Extractsfrom IMMU-132-treated animals also were acid hydrolyzed to release SN-38from the conjugate (i.e., SN-38 (TOTAL]). The results demonstrate thatthe IMMU-132 ADC has the potential to deliver 120 times more SN-38 tothe tumor compared to irinotecan, even though 22-fold less SN-38equivalents were administered with the ADC (not shown).

IMMU-132 Clinical Protocol—

The protocol used in the phase VII study was as indicated in Table 5below.

TABLE 5 Clinical Protocol Using IMMU-132: OVERVIEW Dosing Once weeklyfor 2 weeks administered every 21 days for up to 8 cycles. regimenPatients with objective responses are allowed to continue beyond 8cycles. In the initial enrollment, the planned dose was delayed andreduced if ≥Grade 2 treatment-related toxicity; protocol was amendedlater in study to dose delay and reduction only in the event of ≥Grade 3toxicity. The development of severe toxicities due to treatment requiresdose reduction by 25% of the assigned dose for 1^(st) occurrence, 50%for 2^(nd) occurrence, and treatment discontinued entirely in the eventof a 3^(rd) occurrence. Dose level 8, 12, 18 mg/kg; later reduced to anintermediate dose level of 10 mg/kg. cohorts Cohort size Standard PhaseI [3 + 3] design; expansion includes 15 patients in select cancers. DLTGrade 4 ANC ≥7 d; ≥Grade 3 febrile neutropenia of any duration; Grade 4Platelets ≥5 d; Grade 4 Hgb; Grade 4 N/V/D of any duration or any Grade3 N/V/D for >48 h; Grade 3 infusion-related reactions; ≥Grade 3 non-heme toxicity at least possibly due to study drug. Maximum Maximum dosewhere ≥2/6 patients tolerate the full 21-d treatment cycle Acceptablewithout dose delay or reduction or ≥Grade 3 toxicity. Dose (MAD)Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast, prostate, ovarian, renal, urinarybladder, head and neck, hepatocellular. Refractory/relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Patients were administered IMMU-132 according to the protocol summarizedabove. An exemplary case study is as follows. A 34 y/o white maleinitially diagnosed with metastatic pancreatic cancer (liver) hadprogressed on multiple chemotherapy regimens, includinggemcitabine/Erlotinib/FG-3019, FOLFIRINOX and GTX prior to introductionof IMMU-132 (8 mg/kg dose given days 1 and 8 of a 21 day cycle). Thepatient received the drug for 4 mo with good symptomatic tolerance, animprovement in pain, a 72% maximum decline in CA19-9 (from 15885 U/mL to4418 U/mL) and stable disease by CT RECIST criteria along with evidenceof tumor necrosis. Therapy had to be suspended due to a liver abscess;the patient expired ˜6 weeks later, 6 mo following therapy initiation.

Conclusions

Preclinical studies indicated that IMMU-132 delivers 120-times theamount of SN-38 to a human pancreatic tumor xenograft than whenirinotecan is given. As part of a larger study enrolling patients withdiverse metastatic solid cancers, the Phase 2 dose of IMMU-132 wasdetermined to be 8 to 10 mg/kg, based on manageable neutropenia anddiarrhea as the major side effects. No anti-antibody or anti-SN-38antibodies have been detected to-date, even with repeated therapeuticcycles.

A study of 14 advanced PDC patients who relapsed after a median of 2prior therapies showed CT-confirmed antitumor activity consisting of8/13 (62%) with stable disease. Median duration of TTP for 13 CTassessable pts was 12.7 weeks compared to 8.0 weeks estimated from lastprior therapy. This ADC, with a known drug of nanomolar toxicity,conjugated to an antibody targeting Trop-2 prevalent on many epithelialcancers, by a linker affording cleavage at the tumor site, represents anew efficacious strategy in pancreatic cancer therapy with ADCs. Incomparison to the present standard of care for pancreatic cancerpatients, the extension of time to progression in pancreatic cancerpatients, particularly in those resistant to multiple prior therapies,was surprising and could not have been predicted.

Example 11. Combining Antibody-Targeted Radiation (Radioimmunotherapy)and Anti-Trop-2-SN-38 ADC Improves Pancreatic Cancer Therapy

We previously reported effective anti-tumor activity in nude micebearing human pancreatic tumors with ⁹⁰Y-humanized PAM4 IgG (hPAM4;⁹⁰Y-clivatuzumab tetraxetan) that was enhanced when combined withgemcitabine (GEM) (Gold et al., Int J. Cancer 109:618-26, 2004; ClinCancer Res 9:3929S-37S, 2003). These studies led to clinical testing offractionated ⁹⁰Y-hPAM4 IgG combined with GEM that is showing encouragingobjective responses. While GEM is known for its radiosensitizingability, alone it is not a very effective therapeutic agent forpancreatic cancer and its dose is limited by hematologic toxicity, whichis also limiting for ⁹⁰Y-hPAM4 IgG.

As discussed in the Examples above, an anti-Trop-2 ADC composed of hRS7IgG linked to SN-38 shows anti-tumor activity in various solid tumors.This ADC is very well tolerated in mice (e.g., ≥60 mg), yet just 4.0 mg(0.5 mg, twice-weekly×4) is significantly therapeutic. Trop-2 is alsoexpressed in most pancreatic cancers.

The present study examined combinations of ⁹⁰Y-hPAM4 IgG withsacituzumab govitecan in nude mice bearing 0.35 cm³ subcutaneousxenografts of the human pancreatic cancer cell line, Capan-1. Mice(n=10) were treated with a single dose of ⁹⁰Y-hPAM4 IgG alone (130 i.e.,the maximum tolerated dose (MTD) or 75 with sacituzumab govitecan alone(as above), or combinations of the 2 agents at the two ⁹⁰Y-hPAM4 doselevels, with the first ADC injection given the same day as the⁹⁰Y-hPAM4. All treatments were tolerated, with ≤15% loss in body weight.Objective responses occurred in most animals, but they were more robustin both of the combination groups as compared to each agent given alone.All animals in the 0.13-mCi ⁹⁰Y-hPAM4 IgG+sacituzumab govitecan groupachieved a tumor-free state within 4 weeks, while other animalscontinued to have evidence of persistent disease. These studies providethe first evidence that combined radioimmunotherapy and ADC enhancesefficacy at safe doses.

In the ongoing PAM4 clinical trials, a four week clinical treatmentcycle is performed. In week 1, subjects are administered a dose of¹¹¹In-hPAM4, followed at least 2 days later by gemcitabine dose. Inweeks 2, 3 and 4, subjects are administered a ⁹⁰Y-hPAM4 dose, followedat least 2 days later by gemcitabine (200 mg/m²). Escalation started at3×6.5 mCi/m². The maximum tolerated dose in front-line pancreatic cancerpatients was 3×15 mCi/m² (hematologic toxicity is dose-limiting). Of 22CT-assessable patients, the disease control rate (CR+PR+SD) was 68%,with 5 (23%) partial responses and 10 (45%) having stabilization as bestresponse by RECIST criteria.

Preparation of Antibody-Drug Conjugate (ADC)

The SN-38 conjugated hRS7 antibody was prepared as described above andaccording to previously described protocols (Moon et al. J Med Chem2008, 51:6916-6926; Govindan et al., Clin Cancer Res 2009.15:6052-6061). A reactive bifunctional derivative of SN-38 (CL2A-SN-38)was prepared. The formula of CL2A-SN-38 is(maleimido-[x]-Lys-PABOCO-20-O—SN-38, where PAB is p-aminobenzyl and ‘x’contains a short PEG). Following reduction of disulfide bonds in theantibody with TCEP, the CL2A-SN-38 was reacted with reduced antibody togenerate the SN-38 conjugated RS7.

⁹⁰Y-hPAM4 is prepared as previously described (Gold et al., Clin CancerRes 2003, 9:3929S-37S; Gold et al., Int J Cancer 2004, 109:618-26).

Combination RAIT+ADC

The Trop-2 antigen is expressed in most epithelial cancers (lung,breast, prostate, ovarian, colorectal, pancreatic) and sacituzumabgovitecan conjugates are being examined in various human cancer-mousexenograft models. Initial clinical trials with ⁹⁰Y-hPAM4 IgG plusradiosensitizing amounts of GEM are encouraging, with evidence of tumorshrinkage or stable disease. However, therapy of pancreatic cancer isvery challenging. Therefore, a combination therapy was examined todetermine whether it would induce a better response. Specifically,administration of sacituzumab govitecan at effective, yet non-toxicdoses was combined with RAIT with ⁹⁰Y-hPAM4 IgG.

The results demonstrated that the combination of sacituzumab govitecanwith ⁹⁰Y-hPAM4 was more effective than either treatment alone, or thesum of the individual treatments (not shown). At a dosage of 75 μCi⁹⁰Y-hPAM4, only 1 of 10 mice was tumor-free after 20 weeks of therapy(not shown), the same as observed with sacituzumab govitecan alone (notshown). However, the combination of sacituzumab govitecan with ⁹⁰Y-hPAM4resulted in 4 of 10 mice that were tumor-free after 20 weeks (notshown), and the remaining subjects showed substantial decrease in tumorvolume compared with either treatment alone (not shown). At 130 μCi⁹⁰Y-hPAM4 the difference was even more striking, with 9 of 10 animalstumor-free in the combined therapy group compared to 5 of 10 in the RAITalone group (not shown). These data demonstrate the synergistic effectof the combination of sacituzumab govitecan with ⁹⁰Y-hPAM4. RAIT+ADCsignificantly improved time to progression and increased the frequencyof tumor-free treatment. The combination of ADC with sacituzumabgovitecan added to the MTD of RAIT with ⁹⁰Y-hPAM4 had minimal additionaltoxicity, indicated by the % weight loss of the animal in response totreatment (not shown).

The effect of different sequential treatments on tumor survivalindicated that the optimal effect is obtained when RAIT is administeredfirst, followed by ADC (not shown). In contrast, when ADC isadministered first followed by RAIT, there is a decrease in theincidence of tumor-free animals (not shown). Neither unconjugated hPAM4nor hRS7 antibodies had anti-tumor activity when given alone (notshown).

Example 12. Use of Sacituzumab Govitecan (IMMU-132) to TreatTherapy-Refractive Metastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ER/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on 09/26/12 showed progression of diseaseon the chest wall and enlarged, solid, axillary nodes. The patient wasgiven oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain MRI was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 13. Use of Sacituzumab Govitecan (IMMU-132) to Treat Refractory,Metastatic, Small-Cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and MRI evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givenIMMU-132 at a dose of 18 mg/kg once every three weeks for a total of 5infusions. After the second dose, she experiences hypotension and aGrade 2 neutropenia, which improve before the next infusion. After thefifth infusion, a CT study shows 13% shrinkage of her target left lungmass. MM of the brain also shows a 10% reduction of this metastasis. Shecontinues her IMMU-132 dosing every 3 weeks for another 3 months, andcontinues to show objective and subjective improvement of her condition,with a 25% reduction of the left lung mass and a 21% reduction of thebrain metastasis.

Example 14. Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with Sacituzumab Govitecan (IMMU-132)

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given therapy with IMMU-132 (hRS7-SN-38) at a doseof 10 mg/kg infused every-other-week for a total of 6 doses, after whichCT studies are done to assess status of his disease. These infusions aretolerated well, with some mild nausea and diarrhea, controlled withsymptomatic medications. The CT studies reveal that the sum of his indexmetastatic lesions has decreased by 28%, so he continues on this therapyfor another 5 courses. Follow-up CT studies show that the diseaseremains about 35% reduced by RECIST criteria from his baselinemeasurements prior to IMMU-132 therapy, and his general condition alsoappears to have improved, with the patient regaining an optimisticattitude toward his disease being under control.

Example 15. Clinical Trials of IMMU-132 in Diverse Trop-2 PositiveCancers

Abstract

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CMS chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT=˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer.

The present Phase I trial evaluated this ADC as a potential therapeuticfor pretreated patients with a variety of metastatic solid cancers. Inparticular embodiments, the therapy is of use to treat patients who hadpreviously been found to be resistant to, or had relapsed from, standardanti-cancer treatments, including but not limited to treatment withirinotecan, the parent compound of SN-38. These results were surprisingand unexpected and could not have been predicted.

Sacituzumab govitecan was administered on days 1 and 8 of 21-day cycles,with cycles repeated until dose-limiting toxicity or progression. Doseescalation followed a standard 3+3 scheme with 4 planned dose levels anddose delay or reduction allowed. Twenty-five patients (52-60 years old,3 median prior chemotherapy regimens) were treated at dose levels of 8(N=7), 10 (N=6), 12 (N=9), and 18 (N=3) mg/kg. Neutropenia wasdose-limiting, with 12 mg/kg the maximum tolerated dose for cycle 1, buttoo toxic with repeated cycles. Lower doses were acceptable for extendedtreatment with no treatment-related grade 4 toxicities and grade 3toxicities limited to fatigue (N=3), neutropenia (N=2), diarrhea (N=1),and leukopenia (N=1). Using CT-based RECIST 1.1 criteria, 3 patientsachieved partial responses (triple-negative breast cancer, small-celllung cancer, colon cancer) and 15 others had stable disease as bestresponse; of these, 12 maintained disease control with continuedtreatment for 16-36 weeks. No pre-selection of patients based on tumorTrop-2 expression was undertaken.

It was concluded that sacituzumab govitecan is a promising ADC conjugatewith acceptable toxicity and encouraging therapeutic activity inpatients with difficult-to-treat cancers. The 8 and 10 mg/kg doses wereselected for Phase II studies.

Introduction

Two new antibody-drug conjugates (ADCs) incorporating differentultratoxic (picomolar potency) drugs have been approved, leading tofurther development of other ADCs based on similar principles, includinguse of ultratoxic drugs (Younes et al., 2011, Nat Rev Drug Discov11:19-20; Sievers & Senter, 2013, Ann Rev Med 64:15-29; Krop & Winer,2014, Clin Cancer Res 20:15-20). Alternatively, Moon et al. (2008, J MedChem 51:6916-26) and Govindan et al. (2009, Clin Cancer Res 15:6052-61)selected SN-38, a topoisomerase I inhibitor that is the activemetabolite of irinotecan, an approved drug with well-known but complexpharmacology (Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).Several linkers for conjugating SN-38 were evaluated for release fromthe IgG at varying rates, from several hours to days (Moon et al., 2008,J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61; Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theoptimal linker that was selected, designated CL2A, exhibiting anintermediate conjugate stability in serum, was attached to the hydroxylgroup on SN-38's lactone ring, thereby protecting this ring from openingto the less toxic carboxylate form while bound to the linker, andcontained a short polyethylene glycol moiety to enhance solubility(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The active form ofSN-38 was liberated when the carbonate bond between the linker and SN-38was cleaved, which occurred at low pH, such as that found in lysosomes,as well as the tumor microenvironment, or possibly through enzymaticdegradation.

The antibody chosen for this ADC targeted a tumor-associated antigen,Trop-2 (trophoblast cell-surface antigen) (Cardillo et al., 2011, ClinCancer Res 17:3157-69), using the humanized RS7 monoclonal antibody thatwas shown previously to internalize (Stein et al., 1993, Int J Cancer55:938-46. Trop-2 is an important tumor target for an ADC, because it isover-expressed on many epithelial tumors, particularly more aggressivetypes (Ambrogi et al., 2014, PLoS One 9:e96993; Cubas et al., 2009,Biochim Biophys Act 1796:309-14; Trerotola et al., 2013, Oncogene32:222-33). Trop-2 is also present on a number of normal tissues, butpreclinical studies in monkeys that express the antigen observed onlydose-limiting neutropenia and diarrhea with this new ADC, with noevidence of appreciable toxicity to the Trop-2-expressing normal tissues(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Therefore, withpreclinical data demonstrating activity in several human tumor xenograftmodels and showing a high therapeutic window (Cardillo et al., 2011,Clin Cancer Res 17:3157-69), a Phase I clinical trial was initiated todetermine the maximum tolerated and optimal doses of this novel ADC inheavily-pretreated patients with diverse, relapsed/refractory,metastatic epithelial tumors. This trial was registered atClinicalTrials.gov (NCT01631552).

Materials and Methods

Entry Criteria—

The primary objective was to determine the safety and tolerability ofsacituzumab govitecan (IMMU-132) as a single agent. The trial wasdesigned as a standard 3+3 Phase I design, starting at a dose of 8 mg/kgper injection, with dosages given weekly for 2 weeks in a 3-weektreatment cycle.

Male and non-pregnant, non-lactating females ≥18 years of age wereeligible if they had a diagnosis of one of thirteen different types ofepithelial tumors. Although no pre-selection based on Trop-2 expressionwas required, these tumors are expected to have Trop-2 expressionin >75% of the cases based on immunohistology studies on archivalspecimens. Patients were required to have measurable metastatic disease(no single lesions ≥5 cm) and had relapsed or were refractory to atleast one approved standard chemotherapeutic regimen for thatindication. Other key criteria included adequate (grade ≤1) hematology,liver and renal function, and no known history of anaphylactic reactionsto irinotecan, or grade ≥3 gastrointestinal toxicity to prior irinotecanor other topoisomerase-I treatments. Since patients with such diversediseases were allowed, prior irinotecan therapy was not a prerequisite.Patients with Gilbert's disease or those who had not toleratedpreviously administered irinotecan or with known CNS metastatic diseasewere excluded.

Study Design—

Baseline evaluations were performed within 4 weeks of the start oftreatment, with regular monitoring of blood counts, serum chemistries,vital signs, and any adverse events. Anti-antibody and anti-SN-38antibody responses were measured by ELISA, with samples taken atbaseline and then prior to the start of every even-numbered treatmentcycle. The first CT examination was obtained 6-8 weeks from the start oftreatment and then continued at 8- to 12-week intervals untilprogression. Additional follow-up was required only to monitor anyongoing treatment-related toxicity. Toxicities were graded using the NCICTCAE version 4.0, and efficacy assessed by RECIST 1.1.

An ELISA to detect Trop-2 in serum was developed that has a sensitivityof 2 ng/mL, but after testing 12 patients and finding no evidence ofcirculating Trop-2, no further screening was performed. Although not aneligibility criterion, specimens of previously archived tumors wererequested for Trop-2 determination by immunohistology, using a goatpolyclonal antibody anti-human Trop-2 (R&D Systems, Minneapolis, Minn.),since the epitope recognized by the ADC's antibody, hRS7, is notpreserved in formalin-fixed, paraffin-embedded sections (Stein et al.,1993, Int J Cancer 55:938-46). Staining was performed as describedbelow.

Therapeutic Regimen—

Lyophilized sacituzumab govitecan was reconstituted in saline andinfused over 2-3 h (100 mg of antibody contained ˜1.6 mg of SN-38, witha mean drug:antibody ratio [DAR] of 7.6:1). Prior to the start of eachinfusion, most patients received acetaminophen, anti-histamines (H1 andH2 blockers), and dexamethasone. Prophylactic use of antiemetics oranti-diarrheal medications was prohibited. Therapy consisted of 2consecutive doses given on days 1 and 8 of a 3-week treatment cycle,with the intent to allow patients to continue treatment for up to 8cycles (i.e., 16 treatments) unless there was unacceptable toxicity orprogression. Patients showing disease stabilization or response after 8cycles could continue treatments.

Dose-limiting toxicities (DLT) were considered as grade ≥3 febrileneutropenia of any duration, grade 3 thrombocytopenia with significantbleeding or grade 4 thrombocytopenia ≥5 days, any grade 3 nausea,vomiting or diarrhea that persisted for >48 h despite optimal medicalmanagement, or grade 4 (life threatening) nausea, vomiting or diarrheaof any duration, or any other grade ≥3 non-hematologic toxicity at leastpossibly due to study drug, as well as the occurrence of any grade 3infusion-related reactions.

The maximum tolerated dose (MTD) was judged on the patient's toleranceto the first treatment cycle. On a scheduled treatment day, any patientwith grade ≥2 treatment-related toxicity, with the exception ofalopecia, had their treatment delayed in weekly increments for up to 2weeks. Treatment was reinitiated once toxicity had resolved to grade ≤1.The protocol also initially required all subsequent treatment doses tobe reduced (25% if recovered within 1 week, 50% if within 2 weeks), butthis was relaxed later in the trial when the protocol was amended topermit supportive care after the first cycle. However, if toxicity didnot recover within 3 weeks or worsened, treatment was terminated.Importantly, a dose delay with reduction did not constitute a DLT, andtherefore this allowed treatments to continue, but at a lower dose.Therefore, a patient requiring a dose delay/reduction who was able tocontinue treatment was not considered assessable for DLT, and thenreplaced.

Since a DLT event resulted in the termination of all further treatments,a secondary objective was to assess a dose level that could be toleratedover multiple cycles of treatment with minimal dose delays orreductions. This dose level was designated the maximum acceptable dose,and required patients to tolerate a given dose level in the first cyclewithout having a delay or reduction during that cycle and leading up tothe start of the second cycle.

Pharmacokinetics and Immunogenicity—

Blood samples were taken within ˜30 min from the end of the infusion(e.g., peak) and then prior to each subsequent injection (e.g., trough).Samples were separated and sera frozen for determination of total IgGand sacituzumab govitecan concentrations by ELISA. Serum samples fromseven patients also were assayed for SN-38 content, both total(representing SN-38 bound to the IgG and free) and free SN-38 (i.e.,unbound SN-38).

Results

Patient Characteristics—

Twenty-five patients were enrolled (Table 6). The median age ranged from52 to 60 years, with 76% having an ECOG 1 performance status, theremaining ECOG 0. Most patients had metastatic pancreatic cancer (PDC)(N=7), followed by triple-negative breast cancer (TNBC) (N=4),colorectal cancer (CRC) (N=3), small cell lung cancer (SCLC) (N=2), andgastric cancer (GC) (N=2), with single cases of esophagealadenocarcinoma (EAC), hormone-refractory prostate cancer (HRPC),non-small cell lung cancer (NSCLC), epithelial ovarian cancer (EOC),renal, tonsil, and urinary bladder cancers (UBC).

Immunohistology was performed on archival tissues from 17 patients, with13 (76.4%) having 2+ to 3+ membrane and cytoplasmic staining on >10% ofthe tumor cells in the specimens; 3 specimens (17.6%) were negative.Several representative cases are disclosed below.

All patients entered the trial with metastatic disease in sites typicalfor their primary cancer. CT determined that the median sum of thelargest tumor diameters for all patients was 9.7 cm (range 2.9 to 29.8cm), with 14 patients having 3 or more target lesions (over all patientsmedian=4, range 1-10 lesions) and a median of 2 non-target lesions(range=0-7 lesions) identified in their baseline studies. The mediannumber of prior systemic therapies was 3, with 7 patients (2 PDC and GC,1 each CRC, TNBC, tonsil) having one prior therapy, and 7 having five ormore prior therapies; eleven patients had prior radiation therapy. Priortopoisomerase I therapy was given to nine patients, with 2/3 CRC, 4/7PDC, and 1 patient with EAC receiving irinotecan, and 2/2 patients withSCLC having topotecan, with three of these (2 with SCLC and one withCRC) failing to respond to the anti-topoisomerase 1 therapy. Further,seven of 23 patients (2 undetermined) had responded to their last priortherapy, with a median duration of 3 months (range, 1-11 months).

Nearly all patients received multiple sacituzumab govitecan treatments(median, 10 doses) until there was definitive evidence of diseaseprogression by CT using RECIST 1.1; one patient withdrew becausesystematic deterioration, and 1 patient did not have their target lesionmeasured in first follow-up when a new lesion was observed.

TABLE 6 Baseline demographics and disease characteristics (N = 25patients). 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg M/F 2/5 3/3 3/6 2/1 Age, yMedian (range) 52 (43-62) 58.5 (49-80) 60 (50-74) 56 (52-60) ECOGperformance status 0 3 1 2 0 1 1 5 7 3 Tumor Type N N N N Colorectal 2 10 0 Pancreas 3 1 3 0 TNBC 0 1 2 1 SCLC 0 0 1 1 Other^(a) 2 3 3 1 (EOC,(GC, (UBC, (EAC) GC) RCC, NSCLC, Tonsil) HRPC) Trop-2 expression N  1+ 1(TNBC)  2+ 3 (CRC)  3+ 10 (2 each of EAC, PDC, TNBC; 1 each of EOC,Tonsil, NSCLC, SCLC) Negative 3 (1 each of TNBC, Gastric, Renal) Notdetermined 8 (5 PDC, 1 each of HRPC, SCLC, UBC) Prior Therapy N N N NRadiotherapy 2 4 3 2 Systemic therapy^(c) 1 4 2 1 0 2 0 1 3 1 3 1 1 0 14 1 0 2 0 ≥5  1 2 3 1 Prior Topoisomerase I inhibitor 3 1 4 1 Tumormetastases (# patients) N N N N Target and non-target sitesChest/head/neck 0 2 4 3 Liver^(b) 4 (3) 4 (2) 5 (3) 2 (1) Lungs^(b) 4(3) 4 (2) 4 (3) 1 (1) Lymph nodes 3 2 5 2 Abdomen/pelvis 4 3 4 2 Bone 12 2 1 ≥3 target lesions 4 5 5 0 Patients treated 7 6 9 3Delay/adjustment 1^(st) cycle 1 0 5 2 Dose-limiting toxicity 1^(st)cycle 0 0 0 2 # treatments at this dose median (range) 3 (1-31) 10(1-31) 3 (1-8) 1 (1-2) Total # treatments median (range) 6 (3-31) 10(1-31) 12 (4-34) 4 (3-16) ^(a)Other cancers include ovarian (EOC),gastric (GC), urinary bladder (UBC), non-small cell lung cancer (NSCLC),hormone refractory prostate cancer (HRPC), esophageal adenocarcinoma(EAC), renal cell cancer (RCC), and a squamous cell carcinoma of thetonsil. ^(b)Number of patients with liver or lung involvement; inparenthesis number of these patients with both liver and lunginvolvement. ^(b)Systemic therapy includes chemotherapy and other formsof therapy, including biologicals and investigational agents.

Dose Assessment—

There were no dose delays or reductions, nor DLT events in the 3patients (1 CRC, 2 PDC) enrolled at the starting dose level of 8.0mg/kg. At the next dose level of 12 mg/kg, nine patients were enrolledbecause of protocol-required delays in administering the second dosewere encountered. Five patients experienced a delay in the first cycle(4 had a 1-week delay, with 2 given myeloid growth factor support, and 1patient having a 2-week delay before being given a second dose). All but1 of these patients received 12 mg/kg as their second dose. Four of thenine patients at the 12 mg/kg dose level had their third dose thatstarted the second cycle decreased to 9 mg/kg, and the second cycle wasdelayed 1 additional week in 3 patients. Despite these protocol-requireddelays/reductions, none of the 9 patients had a dose-limiting eventduring the first cycle (e.g., 1 patient had disease-related grade 3hemoglobin after first dose, 2 patients with grade 3 neutropenia afterfirst dose were given myeloid growth factors, 1 had grade 3 neutropeniaafter first dose that recovered without support, 2 had grade 3neutropenia after second dose, 2 patients had grade 2 neutropenia afterthe first or second dose, and 1 patient had no adverse events), andtherefore accrual to the 18 mg/kg dose level was allowed. Here, allthree patients had dose delays after their first treatment, with only 1patient receiving the second treatment at 18 mg/kg. Two patients haddose-limiting grade 4 neutropenia, 1 after first dose, the other afterthe second 18 mg/kg dose, with this latter patient also experiencinggrade 2 diarrhea after this dose. Therefore, with 0/9 patients havingDLT in the first cycle at 12 mg/kg, this level was declared the MTD.

Additional dose-finding studies continued to refine the dose level thatwould allow multiple cycles to be given with minimal delay betweentreatments/cycles. Therefore, 4 more patients were enrolled at the 8mg/kg dose level, and a new intermediate level of 10 mg/kg was opened.Of the initial three patients enrolled at 8 mg/kg, two CRC patientscontinued treatment at 8 mg/kg for a total of 31 and 11 treatments,while a PDC patient received three 8 mg/kg doses before dose reductionto 6 mg/kg because of a grade-2 neutropenia on the fourth dose, and thencompleted 3 more treatments at this level before withdrawing due todisease progression. The additional 4 patients received 3 to 9 doses of8 mg/kg before withdrawing with disease progression. Two of thesepatients received only 1 dose before a protocol-required reduction to 6mg/kg, because of a grade-2 rash and grade-2 neutropenia.

Five of the six patients enrolled at 10 mg/kg received 6 to 30 doseswithout reduction before withdrawing due to disease progression. One GCpatient (#9) developed grade 3 febrile neutropenia as well as grade 4hemoglobin after receiving 1 dose. While the febrile neutropenia wasconsidered possibly-related to treatment, because it occurred shortlyafter the first dose, a perforation in the stomach lining was found tolikely contribute to the grade 4 hemoglobin, and was consideredunrelated. Ultimately, the patient had rapid deterioration and died 4weeks from the first dose.

Thus, while the overall results supported 12 mg/kg as the MTD, since 8to 10 mg/kg were better tolerated in the first cycle and permittedrepeated cycles with minimal toxicity, Phase II clinical studies are inprogress to evaluate these 2 dose levels.

Adverse Events—

There were 297 infusion of sacituzumab govitecan given over 2-3 h, withmost investigators electing to pre-medicate prior to each infusion.There were no infusion-related adverse events. While more than half ofthe patients experienced fatigue, nausea, alopecia, diarrhea, andneutropenia that were considered at least likely related to sacituzumabgovitecan treatment; these were mostly grade 1 and 2 (not shown). Themost reported grade 3 or 4 toxicity was neutropenia (N=8), but six ofthese patients were treated initially at 12 and 18 mg/kg. Febrileneutropenia occurred in 2 patients, one was the GC patient #9 alreadymentioned who received only one 10 mg/kg dose, and a second PDC patient(#19), who had received 4 doses of 12 mg/kg. Diarrhea was mild in mostpatients, with only three (12%) experiencing grade 3. Two occurred atthe 12 mg/kg dose level, 1 after receiving 4 doses, and the other afterthe first dose, but this patient received 6 more doses at 12 mg/kg withonly grade 2 diarrhea reported. Subsequently, both patients wereprescribed an over-the-counter anti-diarrheal and treatment continued.There were no other significant toxicities associated with sacituzumabgovitecan, but two patients reported a grade 2 rash and 3 patients had agrade 1 pruritus.

Efficacy—

The best response was measured by the change in target lesions andtime-to-progression data from patients who had at least onepost-treatment CT measurement of their target lesions (not shown). Fourpatients with disease progression are not represented in the graph,because they did not have a follow-up CT assessment (N=1) or they hadnew lesions and therefore progressed irrespective of their target lesionstatus (N=3). Overall, 3 patients had more than a 30% reduction in theirtarget lesions (partial response, PR). Two of these patients (#3 and#15) had confirmatory follow-up CTs, while the third patient (#22)progressed at the next CT performed 12 weeks later. Fifteen patients hadstable disease (SD), and 7 progressed (PD) as the best response byRECIST 1.1. The median time to progression from the start of treatmentfor 24 patients (excluding 1 patient who received only 1 treatment andwithdrew) was 3.6 months [range, 1-12.8 months]; 4.1 months (range,2.6-12.8 months) for all patients with SD or PR (N=18). Of the ninepatients who received prior therapy containing a topoisomerase-Iinhibitor, two had significant reductions of their target lesions (28%and 38%), 5 had stable disease, including 2 for sustained periods (4.1and 6.9 months, respectively), whereas 2 progressed at their firstassessment.

Comparing TTP with survival of these patients indicates that 16 patientssurvived from onset of therapy for 15-20 months, including two with a PR(patients 15 (TNBC) and 3 (CRC), and the other four with SD (2 CRC, 1HRPC, 1 TNBC) (not shown). Radiological responses included 2 patientswith >30% reduction in their target lesions (PR) (not shown).

In addition to the 3 patients with PR as best response, there wereseveral notable cases of extended stable disease. A 50-year-old patientwith TNBC (patient 18; immunohistology Trop-2 expression=3+) experienceda 13% reduction after just 3 doses, culminating after 16 doses in a 19%reduction in the 4 target lesions (SLD decreased from 7.5 to 6.1 cm),before progressing 45 weeks after starting treatment and receiving 26doses. A 63-year-old female with CRC (patient 10; immunohistology 2+)with 7 prior treatments, including 3 separate courses of anirinotecan-containing regimens, had an overall 23% reduction in 5 targetlesions after receiving 5 doses of 10 mg/kg sacituzumab govitecan,culminating in a maximum 28% reduction after 18 doses. Her plasma CEAdecreased to 1.6 ng/mL from a baseline level of 38.5 ng/mL. Afterreceiving 25 doses (27 weeks), she had PD with a 20% increase from theSLD nadir. Interesting, plasma CEA at the time treatment ended was only4.5 ng/mL. A 68-year-old patient with HRPC (patient 20; noimmunohistology) presented with 5 target lesions (13.3 cm) and 5non-target lesions (3 bone metastases). He received 34 treatments over aperiod of 12.7 months until progression, with PSA levels increasinggradually over this time. Another notable case was a 52-year-old malewith esophageal cancer (patient 25; immunohistology 3+) who had received6 prior therapies, including 6 months of FOLFIRI as his 3^(rd) course oftreatment. Treatment was initiated at 18 mg/kg of sacituzumab govitecan,which was reduced to 13.5 mg/kg because of neutropenia. He had SD over aperiod of 30 weeks, receiving 15 doses before progressing. A 60-year-oldfemale with PDC (#11) with liver metastases was treated at 10 mg/kg. Herbaseline CA19-9 serum titer decreased from 5880 to 2840 units/mL after 8doses and there was disease stabilization (12% shrinkage as bestresponse) for a period or 15 weeks (11 doses) before a new lesion wasdiscovered. Nevertheless, because CA19-9 remained reduced (2814units/mL), the patient received another 8 treatments (3 months) at 10mg/kg before coming off study with progression of her target lesions.

At this time, the potential utility of testing Trop-2 expression inarchived samples from this small sampling of 16 patients with diversecancers is insufficient to allow for a definitive assessment, primarilybecause most showed elevated expression.

PK and Immunogenicity—

Concentrations of sacituzumab govitecan and IgG in the 30-min serumsample are provided in Table 7, which showed a general trend for thevalues to increase as the dose increased. In a representative case, apatient with TNBC (#15) received multiple doses, starting at 12 mg/kg,with subsequent reductions over the course of her treatment.Concentrations of the IgG and sacituzumab govitecan in the 30-min serumover multiple doses by ELISA were similar over time (not shown),adjusting lower when the dose was reduced. While residual IgG could befound in the serum drawn immediately before the next dose (troughsamples), no sacituzumab govitecan could be detected (not shown).

Total SN-38 concentration in the 30-min serum sample of patient 15 was3,930 ng/mL after the first dose in cycle 1 (C1D1), but when sacituzumabgovitecan treatment was reduced to 9.0 mg/kg for the second dose of thefirst cycle (C1D2), the level decreased to 2,947 ng/mL (not shown). Afurther reduction to 2,381 ng/mL was observed in the 6^(th) cycle, whenthe dose was further reduced to 6.0 mg/kg. The amount of free SN-38 inthese samples ranged from 88 to 102 ng/mL (2.4% to 3.6% of total SN-38),illustrating that >96% of the SN-38 in the serum in these peak sampleswas bound to IgG. Twenty-eight 30-min serum samples from 7 patients wereanalyzed by HPLC, with free SN-38 averaging 2.91±0.91% of the totalSN-38 in these samples. Free SN-38G concentrations measured in 4patients never exceeded SN-38 levels, and were usually several-foldlower. For example, patient #25 had determinations assessed in the30-min sample for 12 injections over 8 cycles of treatment. At astarting dose of 18 mg/kg, he had 5,089 ng/mL of SN-38 in theacid-hydrolyzed sample (total SN-38) and just 155.2 ng/mL in thenon-hydrolyzed sample (free SN-38; 3.0%). Free SN-38G (glucuronidatedform) in this sample was 26.2 ng/mL, or just 14.4% of the total unboundSN-38+SN-38G in the sample. The patient continued treatment at 13.5mg/kg, with SN-38 averaging 3309.8±601.8 ng/mL in the 11 remaining peak,acid-hydrolyzed samples, while free SN-38 averaged 105.4±47.7 ng/mL(i.e., 96.8% bound to the IgG), and free SN-38G averaging 13.9±4.1 ng/mL(11.6% of the total SN-38+SN-38G). Importantly, in nearly all of thepatients, concentrations of SN-38G in the acid-hydrolyzed andnon-hydrolyzed samples were similar, indicating that none of the SN-38bound to the conjugate was glucuronidated.

TABLE 7 Serum concentration (μg/mL) of intact sacituzumab govitecan(ADC) and hRS7 IgG by ELISA. Assays were performed in samples taken 0.5h after the first dose. 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg N 7 5 9 3 IgGADC IgG ADC IgG ADC IgG ADC Mean 193.1 141.5 203.35 185.77 239.2 183.3409.16 258.27 SD 56.5 23.8 55.72 54.14 70.7 71.8 88.78 143.26 Min 96.095.0 152.00 144.00 168.9 98.0 321.48 162.80 Max 249.0 169.3 285.85237.39 400.0 311.0 499.00 423.00

None of these patients had a positive baseline level (i.e., >50 ng/mL)or a positive antibody response to either the IgG or SN-38 over theircourse of treatment.

Discussion

Trop-2 is expressed abundantly in many epithelial tumors, making it anantigen of interest for targeted therapies (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14), especially since it is considered aprognostic marker and oncogene in several cancer types (Cardillo et al.,2011, Clin Cancer Res 17:3157-69; Ambrogi et al., 2014, PLoS One9:e96993; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14;Trerotola et al., 2013, Oncogene 32:222-33). Although its expression innormal tissues and relationship to another well-studied tumor-associatedantigen, EpCam, drew some initial words of caution regarding the safetyof developing immunotherapeutics to Trop-2 (Trerotola et al., 2009,Biochim Biophys Acta 1805:119-20), our studies in Cynomolgus monkeysthat express Trop-2 in tissues similar to humans indicated sacituzumabgovitecan was very well tolerated at a human equivalent dose of ˜40mg/kg (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). At higherdoses, animals experienced neutropenia and diarrhea, known side-effectsassociated with SN-38 derived from irinotecan therapy, yet evidence forsignificant histopathological changes in Trop-2-expressing normaltissues was lacking (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Thus, with other preclinical studies finding sacituzumab govitecan waspotent at the low nanomolar level and effective in a variety of humanepithelial tumor xenografts at non-toxic doses, a phase I trial wasundertaken in patients who had failed one or more standard therapies fortheir diverse metastatic epithelial tumors.

A major finding of this study was that despite using a more conventionaldrug that is not considered as ultratoxic (drugs active in picomolarrange, whereas SN-38 has potency in the low nanomolar range), thesacituzumab govitecan anti-Trop-2-SN-38 conjugate proved clinically tobe therapeutically active in a wide range of solid cancers at doses withmoderate and manageable toxicity, thus exhibiting a high therapeuticindex. A total of 297 doses of sacituzumab govitecan were given to 25patients without incident; 4 patients received >25 injections.Importantly, no antibody response to the hRS7 IgG or SN-38 was detected,even in patients with multiple cycles of treatment for up to 12 months.Although Trop-2 is expressed in low quantities in a variety of normaltissues (Cardillo et al., 2011, Clin Cancer Res 17:3157-69), neutropeniawas the only dose-limiting toxicity, with myeloid growth factor supportused in 2 patients given ≥12 mg/kg of sacituzumab govitecan to expediterecovery and allow continuation of treatment in patients who hadexhausted their options for other therapy. While the MTD was declared tobe 12 mg/kg, 8.0 and 10.0 mg/kg dose levels were selected for furtherexpansion, since patients were more likely to tolerate additional cyclesat these levels with minimal supportive care, and responses wereobserved at these levels. Only 2 of 13 patients (15.4%) experiencedgrade-3 neutropenia at these dose levels. The grade 3 and 4 neutropeniaincidence for irinotecan monotherapy given weekly or once every 3 weeksin a front- or second-line setting was 14 to 26% (Camptosar—irinotecanhydrochloride injection, solution (prescribing information, packageinsert) Pfizer, 2012). With sacituzumab govitecan, only 1 patient at the10 mg/kg dose level had grade-3 diarrhea. This incidence is lower thanthe 31% of patients given weekly×4 doses of irinotecan who experiencedgrades 3 and 4 late diarrhea (Camptosar—irinotecan hydrochlorideinjection, solution (prescribing information, package insert) Pfizer,2012). Other common toxicities attributed to sacituzumab govitecanincluded fatigue, nausea, and vomiting, most being grade 1 and 2, aswell as alopecia. Two incidents of febrile neutropenia and one of grade3 deep vein thrombosis also occurred at the 10 and 12 mg/kg dose levels.UGT1A1 monitoring was not initiated until after dose exploration wascompleted, and therefore an assessment of its contribution to toxicitycannot be reported at this time.

Patients enrolled in this trial were not pre-selected for Trop-2expression, primarily because immunohistological assessments of tissuemicroarrays of diverse cancers (such as prostate, breast, pancreas,colorectal, and lung cancers) had indicated the antigen was presentin >90% of the specimens (not shown). In addition, Trop-2 was not foundin the sera of 12 patients with diverse metastatic cancers, furthersuggesting that a serum assay would not be useful for patient selection.Although we are attempting to collect archival specimens of the tumorsfrom patients enrolled in the trial, there is insufficient evidence atthis time to suggest patient selection based on immunohistologicalstaining will correlate with anti-tumor activity, so no patientenrichment based on Trop-2 expression has been undertaken.

As a monotherapy, sacituzumab govitecan had good anti-tumor activity inpatients with diverse metastatic, relapsed/refractory, epithelialtumors, showing appreciable reductions in target lesions by CT, usingRECIST1.1 criteria, including sustained disease stabilization. Three(12%) of the 25 patients (1 each of SCLC [after progressing withtopotecan], TNBC, and colon cancer) had >30% reductions of their targetlesions before progressing 2.9, 4.3, and 7.1 months, respectively, fromthe onset of therapy. Fifteen patients (60%) had SD, with 9 of theseprogressing after >4 months from the start of treatment. Responses ordisease stabilization occurred in 7 of 9 patients who had prior therapywith a topoisomerase I inhibitor-containing drug or regimen. Three ofthese failed to respond to their prior topoisomerase I inhibitor therapy(irinotecan or topotecan), yet sacituzumab govitecan was able to inducetumor shrinkage in 2 of them: 13% in a patient with colon cancer and 38%in the other with SCLC. Thus, sacituzumab govitecan may betherapeutically active in those who failed or relapsed to a priortopoisomerase I-containing regimen, which should be examined further inthe Phase II expansion study.

Although the largest number of patients enrolled in this trial hadadvanced pancreatic ductal cancer (N=7; median time to progression 2.9months]; range, 1.0 to 4.0 months), even in this difficult-to-treatdisease, there have been encouraging reductions in target lesions andCA19-9 serum concentrations to suggest activity (Picozzi et al., 2014,presented at the AACR Special Conference “Pancreatic Cancer: Innovationsin Research and Treatment, New Orleans, La. USA, p. B99). However,responses in patients with TNBC and SCLC are of particular interest,given the need for targeted therapies in these indications. Indeed,additional partial responses in patients with TNBC (Goldenberg et al.,2014, presented at the AACR San Antonio Breast cancer Symposium, SanAntonio, Tex.) and SCLC (Goldenberg et al., 2014, Sci Transl Med)observed in the on-going expansion phase of this trial have suggestedfurther emphasis on these cancers, but encouraging responses in NSCLC,EAC, UBC, and CRC are also being followed. Indeed, in a recent update ofthe on-going trial of sacituzumab govitecan, in 17 TNBC patients studiedto date, an overall response rate (PR) of 29%, with 46% clinical benefitrate (PR+SD≥6 months) has been observed. Long-term survival (15-20months) was observed for almost 25% (6/25) of the patients studied, andincluded 2 with PRs and 4 with SD, including patients with TNBC (N=2),CRC (N=3), and HRPC (N=1).

Analysis of the serum samples 30 min after the end of infusionshowed >96% of the SN-38 was bound to the IgG. More detailedpharmacokinetics will be available when the phase II portion of thetrial is completed. HPLC analysis also detected only trace amounts offree SN-38G in the serum, whereas with irinotecan therapy the AUC forthe less active SN-38G is >4.5-fold higher than SN-38 (Xie et al., 2002,J Clin Oncol 20:3293-301). Comparison of SN-38 delivery in tumor-bearinganimals given sacituzumab govitecan and irinotecan has indicated theSN-38 bound to the IgG is not glucuronidated, whereas in animals givenirinotecan, >50% of the total SN-38 in the serum is glucuronidated(Goldenberg et al., 2014, J Clin Oncol 32: Abstract 3107). Moreimportantly, analysis of SN-38 concentrations were ˜135-fold higher inCapan-1 human pancreatic cancer xenografts given sacituzumab govitecanthan irinotecan (Goldenberg et al., 2014, Sci Transl Med). Thus,sacituzumab govitecan has several distinct advantage over non-targetedforms of topoisomerase-I inhibitors: (i) a mechanism that selectivelyretains the conjugate in the tumor (anti-Trop-2 binding), and (ii) thetargeted SN-38 also appears to be fully protected (i.e., notglucuronidated and in the lactone form), such that any SN-38 accreted bythe tumor cells either by the direct internalization of the conjugate orthrough its release into the tumor microenvironment from the conjugatebound to the tumor will be in its most potent form. These resultssuggest that a moderately-toxic, but well understood, cytotoxic agent,SN-38, can be effective as part of a tumor-targeting ADC, such assacituzumab govitecan. But by administering an ADC with amoderately-toxic drug conjugated at a high drug:antibody ratio (7.6:1),higher concentrations of SN-38 can be delivered to the cancers targeted,as suggested in the improved concentration of SN-38 achieved withsacituzumab govitecan compared to that released from irinotecan.

In conclusion, this phase I experience has shown that sacituzumabgovitecan was tolerated with moderate and manageable toxicity, allrelated to the activity of SN-38, with no evidence of damage to normaltissues known to contain Trop-2. Importantly, sacituzumab govitecan wasactive in patients with diverse metastatic solid tumors, even afterfailing prior therapy with topoisomerase-I inhibitors. Thus, it appearsfrom this initial experience that sacituzumab govitecan has a hightherapeutic index, even in patients with tumors not known to beresponsive to topoisomerase I inhibitors, such as SCLC and TNBC. Thisclinical trial is continuing, focusing on starting doses of 8 and 10mg/kg in patients with TNBC, SCLC, and other Trop-2⁺ cancers.

Example 16. Use of IMMU-132 in Triple Negative Breast Cancer (TNBC)

The Trop-2/TACSTD2 gene has been cloned (Fornaro et al., 1995, Int JCancer 62:610-18) and found to encode a transmembrane Ca⁺⁺-signaltransducer (Basu et al., 1995, Int J Cancer 62:472-72; Ripani et al.,1998, Int J Cancer 76:671-76) functionally linked to cell migration andanchorage-independent growth, with higher expression in a variety ofhuman epithelial cancers, including breast, lung, gastric, colorectal,pancreatic, prostatic, cervical, head-and-neck, and ovarian carcinomas,compared to normal tissues (Cardillo et al., 2011, Clin Cancer Res17:3157-69; Stein et al., 1994; Int J Cancer Suppl 8:98-102; Cubas etal., 2009, Biochim Biophys Acta 196:309-14; Trerotola et al., 2013,Oncogene 32:222-33). The increased expression of Trop-2 has beenreported to be necessary and sufficient for stimulation of cancer growth(Trerotola et al., 2013, Oncogene 32:222-33), while a bi-cistroniccyclin D1-Trop-2 mRNA chimera is an oncogene (Guerra et al., 2008,Cancer Res 68:8113-21). Importantly, elevated expression has beenassociated with more aggressive disease and a poor prognosis in severalcancer types (Cubas et al., 2009, Biochim Biophys Acta 196:309-14;Guerra et al., 2008, Cancer Res 68:8113-21; Bignotti et al., 2010, Eur JCancer 46:944-53; Fang et al., 2009, Int J Colorectal Dis 24:875-84;Muhlmann et al., 2009, J Clin Pathol 62:152-58), including breast cancer(Ambrogi et al., 2014, PLoS One 9:e96993; Lin et al., 2013, Exp MolPathol 94:73-8). Increased Trop-2 mRNA is a strong predictor of poorsurvival and lymph node metastasis in patients with invasive ductalbreast cancers, and Kaplan-Meier survival curves showed that breastcancer patients with high Trop-2 expression had a significantly shortersurvival (Lin et al., 2013, Exp Mol Pathol 94:73-8).

Methods

DAR Determination by HIC—

Clinical lots of IMMU-132 were analyzed by hydrophobic interactionchromatography (HIC) using a butyl-NPR HPLC column (Tosoh Bioscience,King of Prussia, Pa.). IMMU-132 injections (100 μg) were resolved with a15-min linear gradient of 2.25-1.5 M NaCl in 25 mM sodium phosphate, pH7.4, run at 1 mL/min and room temperature.

DAR Determination by LC-MS—

Because the interchain disulfides are reduced and the resultingsulfhydryl groups are used for drug conjugation (or blocked), the heavyand light chains resolved during LC-MS analysis without addition ofreducing agents, and were analyzed independently. Different lots ofIMMU-132 were injected on an Agilent 1200 series HPLC using an AerisWidepore C4 reverse-phase HPLC column (3.6 50×2.1 mm) and resolved byreverse phase HPLC with a 14-min linear gradient of 30-80% acetonitrilein 0.1% formic acid. Electrospray ionization time of flight (ESI-TOF)mass spectrometry was accomplished with an in-line Agilent 6210 ESI-TOFmass spectrometer with Vcap, fragmentor and skimmer set to 5000V, 300Vand 80V, respectively. The entire RP-HPLC peak representing all kappa orheavy chain species were used to generate deconvoluted mass spectra.

Cell Lines—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection (Manassas, Va.), except where noted,and all were authenticated by short tandem repeat (STR) assay by theATCC.

Trop-2 Surface Expression on Various Human Breast Carcinoma Cell Lines—

Expression of Trop-2 on the cell surface is based on flow cytometry.Briefly, cells were harvested with Accutase Cell Detachment Solution(Becton Dickinson (BD); Franklin Lakes, N.J.; Cat. No. 561527) andassayed for Trop-2 expression using QuantiBRITE PE beads (BD Cat. No.340495) and a PE-conjugated anti-Trop-2 antibody (eBiosciences, Cat. No.12-6024) following the manufacturer's instructions. Data were acquiredon a FACSCalibur Flow Cytometer (BD) with CellQuest Pro software, withanalysis using Flowjo software (Tree Star; Ashland Oreg.).

In Vitro Cytotoxicity Testing—

Sensitivity to SN-38 was determined using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumdye reduction assay (MTS dye reduction assay; Promega, Madison, Wis.).Briefly, cells were plated into 96-well clear, flat-bottomed plates asdescribed above. SN-38 dissolved in DMSO was diluted with media to afinal concentration of 0.004 to 250 nM. Plates were incubated inhumidified chamber for 96 h 37° C./5% CO₂, after which the MTS dye wasadded and placed back into the incubator until untreated control cellshad an absorbance greater than 1.0. Growth inhibition was measured as apercent of growth relative to untreated cells. Dose-response curves weregenerated from the mean of triplicate determinations, and IC₅₀-valueswere calculated using Prism GraphPad Software.

In Vitro Specificity Testing by Flow Cytometry with rH2AX-Stained Cells—

For drug activity testing, HCC1806 and HCC1395 TNBC cell lines cellswere seeded in 6-well plates at 5×10⁵ cells/well and held at 37° C.overnight. After cooling the cells for 10 min on ice, the cells wereincubated with either IMMU-132 or hA20 anti-CD20-5N38 at ˜20 μg/ml(equal SN38/well for both agents) for 30 minutes on ice, washed threetimes with fresh media, and then returned to 37° C. overnight. Cellswere trypsinized briefly, pelleted by centrifugation, fixed in 4%formalin for 15 min, then washed and permeabilized in 0.15% Triton-X100in PBS for another 15 min. After washing twice with 1% bovine serumalbumin-PBS, cells were incubated with mouse anti-rH2AX-AF488 (EMDMillipore Corporation, Temecula, Calif.) for 45 minutes at 4° C. Thesignal intensity of rH2AX was measured by flow cytometry using a BDFACSCalibur (BD Biosciences, San Jose, Calif.).

IHC of Trop-2 in Tumor Microarrays and Patient Specimens—

This involved standard IHC methods on tissue and microarray sections.Scoring was based on the intensity of the stain in >10% of the tumorcells within the specimen, including negative, 1+(weak), 2+(moderate),and 3+(strong).

In Vivo Therapeutic Studies in Xenograft Models—

SN-38 equivalents in a dose of 250 μg ADC to a 20-gram mouse (12.5mg/kg) is equal to 0.2 mg SN-38/kg. For irinotecan (irinotecan-HClinjection; AREVA Pharmaceuticals, Inc., Elizabethtown, Ky.), 10 mgirinotecan/kg converts to 5.8 mg SN-38/kg based on mass.

Immunoblotting—

Cells (2×10⁶) were plated in 6-well plates overnight. The following daythey were treated with either SN-38 or IMMU-132 at an SN-38concentration equivalent of 0.4 μg/mL (1 μM) for 24 and 48 h. ParentalhRS7 was used as a control for the ADC.

Quantification of SN-38 in Mice with Human Tumor Xenografts—

Two groups, each with 15 animals bearing subcutaneous implants of thehuman pancreatic carcinoma cell line, were administered eitheririnotecan or IMMU-132. At 5 different intervals, 3 animals per groupwere euthanized. The Capan-1 tumors (0.131±0.054 g; N=30) were removedand homogenized in deionized water (DI) (1 part tissue+10 parts DIwater); serum was diluted with an equal part DI water. Serum and tissuehomogenates were extracted and analyzed by reversed-phase HPLC(RP-HPLC). While extracted samples were adequate for detecting productsfrom the irinotecan-treated animals, samples from animals given IMMU-132were split into 2 portions, with one undergoing an acid-hydrolysis stepin order to release all of the SN-38 bound to the IgG, which wouldotherwise go undetected in the extracted samples.

Statistics—

Statistical analyses were performed using GraphPad Prism version 5.00for Windows, GraphPad Software, La Jolla Calif. USA. The specifictesting performed is identified with each study.

Results

SN-18 Structure and Properties—

IMMU-132 utilizes the topoisomerase I inhibitor, SN-38, the watersoluble metabolite of the anticancer camptothecin, irinotecan(7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin),that is therapeutically active in colorectal, lung, cervical, andovarian cancers (Garcia-Carbonero et al., 2002, Clin Cancer Res8:641061). An important advantage for selecting SN-38 is that the drug'sin-vivo pharmacology is well known. Irinotecan must be cleaved byesterases to form SN-38, which is 2-3 orders of magnitude more potentthan irinotecan, with activity in the low nanomolar range (Kawato etal., 1991, Cancer Res 51:4187-91). At physiological pH, camptothecinsexist in an equilibrium comprising the more active lactone form and theless active (10% potency) open carboxylic acid form (Burke & Mi, 1994, JMed Chem 37:40-46).

The design of the SN-38 derivative used in IMMU-132, CL2A-SN-38,addressed multiple challenges in using this drug in the ADC format, andinvolved the following features: (i) A short polyethylene glycol (PEG)moiety was placed in the cross-linker to confer aqueous solubility tothis highly insoluble drug; (ii) a maleimide group was incorporated forfast thiol-maleimide conjugation to mildly reduced antibody, with aspecially-designed synthetic procedure enabling high-yield incorporationof maleimide in the context of assembling the carbonate linkage; (iii) abenzylcarbonate site provided a pH-mediated cleavage site to release thedrug from the linker; and (iv) importantly, the crosslinker was attachedto SN-38's 20-hydroxy position, which kept the lactone ring of the drugfrom opening to the less active carboxylic acid form under physiologicalconditions (Giovanella et al., 2000, Ann NY Acad Sci 922:27-35). Thesynthesis of SN-38 derivatives and the conjugation of CL2A-SN-38 tomildly reduced hRS7 IgG has been described above. The limited reductionprocedure breaks only the interchain disulfide bridges between theheavy-heavy and heavy-light chains, but not the intra-domain disulfides,generating 8 site-specific thiols per antibody molecule. It is thenconjugated to CL2A-SN-38, purified by diafiltration, and lyophilized forstorage. During manufacturing, conditions are adjusted to minimize anyloss of SN-38 from IMMU-132, with the final lyophilized productconsistently having <1% free SN-38 when reconstituted. However, whenplaced in serum and held at 37° C., SN-38 is released from the conjugatewith a half-life of ˜1 day (not shown).

The release of SN-38 appears to be an important feature of IMMU-132,with this type of linker selected based on efficacy studies that testedSN-38 conjugated to a variety of linkers that had different rates ofSN-38 release, from ˜10 h release half-life to being highly stable (Moonet al., 2008 (30, 31). Optimal therapeutic activity was found with aconjugate having an intermediate release rate in serum of ˜2 days. Wesubsequently improved the manufacturing process for this type of linker,designated CL2A, by removing a phenylalanine residue (Cardillo et al.,2011, Clin Cancer Res 17:3157-64, and then again compared the efficacywith that of another stably-linked anti-Trop-2 conjugate (CL2) that wasdesigned to release SN-38 only under lysosomal conditions (i.e., in thepresence of cathepsin B and pH 5.0). In animal models, the anti-Trop-2conjugate prepared with the CL2A linker yielded better therapeuticresponses than when SN-38 was linked stably, indicating that evenantibodies that internalized quickly benefited when SN-38 was allowed tobe released in serum with a half-life of ˜1 day (Govidan et al. 2013,Mol Cancer Ther 12:968-78). Since clinical studies with radiolabeledantibodies have found the antibodies localize in tumors within a fewhours, reaching peak concentrations within 1 day (Sharkey et al., 1995,Cancer Res 55:5935s-45s), selectively enhanced concentrations of SN-38are delivered locally in the tumor through internalization of the intactconjugate, extracellular release of the free drug, or both mechanisms inconcert.

Drug-Antibody Ratio (DAR) Determination.

Five clinical lots of IMMU-132 were evaluated by hydrophobic interactionHPLC (HIC-HPLC), which resolved three peaks representing species withDARs of 6, 7 and 8, with the greatest fraction comprising a DAR=8 (notshown). IMMU-132 was produced consistently by this manufacturingprocess, with an overall DAR (DAR_(AVE)) of 7.60±0.03 among the fiveclinical lots (not shown). HIC-HPLC results were confirmed by liquidchromatography-mass spectrometry (LC-MS) (not shown). The analysisshowed that >99% of the 8 available sulfhydryl groups were coupled withthe CL2A linker, either with or without SN-38. There were nounsubstituted (or N-ethylmaleimide capped) heavy or light chainsdetected. Thus, the difference in DAR among the species results fromSN-38 liberation from the linker during manufacturing and not from alower initial substitution ratio. Once prepared and lyophilized,IMMU-132 has been stable for several years.

Effect of DAR on Pharmacokinetics and Anti-Tumor Efficacy in Mice.

Mice bearing Trop-2⁺ human gastric carcinoma xenografts (NCI-N87) weregiven 2 treatments 7 days apart, each with equal protein (0.5 mg) dosesof IMMU-132 having DARs of 6.89, 3.28, or 1.64 (not shown). Animalstreated with the ADCs having a DAR of 6.89 had a significantly improvedmedian survival time (MST) compared to mice given ADCs with either 3.38or 1.64 DARs (MST=39 days vs. 25 and 21 days, respectively; P<0.0014).There was no difference between groups treated with the 3.28 or 1.64 DARconjugates and the saline control group.

To further elucidate the importance of a higher DAR, mice bearingNCI-N87 gastric tumors were administered 0.5 mg IMMU-132 with a DAR of6.89 twice weekly for two weeks (not shown). Another group receivedtwice the protein (1 mg) dose of an IMMU-132 conjugate with a DAR of3.28. Although both groups received the same total amount of SN-38 (36μg) with each dosing scheme, those treated with the 6.89 DAR conjugateinhibited tumor growth significantly more than tumor-bearing animalstreated with the 3.28 DAR conjugate (P=0.0227; AUC). Additionally,treatment with the lower DAR was not significantly different than theuntreated controls. Collectively, these studies indicate that a lowerDAR reduces efficacy.

An examination of the pharmacokinetic behavior of conjugates prepared atthese different ratios was performed in non-tumor-bearing mice given 0.2mg of each conjugate, unconjugated hRS7 IgG, or hRS7 IgG that wasreduced and then capped with N-ethylmaleimide. Serum was taken at 5intervals from 0.5 to 168 h and assayed by ELISA for hRS7 IgG. There wasno significant difference in the clearance of these conjugates comparedto the unconjugated IgG (not shown). Thus, the substitution level didnot affect the pharmacokinetics of the conjugates, and equallyimportant, the reduction of the interchain disulfide bonds did notappear to destabilize the antibody.

Trop-2 Expression in TNBC and SN-38 Sensitivity.

Trop-2 expression was determined by immunohistochemistry (IHC) inseveral tissue microarrays of human tumor specimens. In one microarraycontaining 31 TNBC specimens, as well as 15 hormone-receptor- orHER-2-positive breast cancers, positive staining occurred in over 95% ofthe tumors, with 3+ staining indicated in 65% of the cases.

Table 8 lists 6 human breast cancer cell lines, including four TNBC,showing their surface expression of Trop-2 and sensitivity to SN-38.Trop-2 surface expression in 5 of the 6 cell lines exceeded 90,000copies per cell. SN-38 potency ranged from 2 to 6 nM in 5 of the 6 celllines, with MCF-7 having the lowest sensitivity of 33 nM. In vitropotency for IMMU-132 is not provided, because nearly all of the SN-38associated with IMMU-132 is released into the media during the 4-dayincubation period, and therefore its potency would be similar to that ofSN-38. Therefore, a different strategy was required to illustrate theimportance of antibody targeting as a mechanism for delivering SN-38.

TABLE 8 Trop-2 expression and SN-38 sensitivity in breast cancer celllines. Receptor Trop-2 surface IC₅₀ (nM) Cell Line status expression^(A)SN-38 SK-BR-3 HER2⁺ 328,281 ± 47,996 2 MDA-MB-468 TNBC 301,603 ± 29,4702 HCC38 TNBC 181,488 ± 69,351 2 MCF-7 ER⁺ 110,646 ± 17,233 33 HCC1806TNBC  91,403 ± 20,817 1 MDA-MB-231 TNBC 32,380 ± 5,460 6 ^(A)Mean ± SDnumber of surface Trop-2 molecules per cell from three separate assays.

Antigen-positive (HCC1806) or -negative (HCC1395) TNBC cell lines thatwere incubated at 4° C. for 30 min with either IMMU-132 or a non-bindinganti-CD20 SN-38 conjugate. The cells were then washed to remove unboundconjugate, and then incubated overnight at 37° C. Cells were fixed andpermeabilized, and then stained with the fluorescentanti-phospho-histone H2A.X antibody to detect dsDNA breaks by flowcytometry (Bonner et al., 2008, Nat Rev Cancer 8:957-67) (Table 9). TheTrop-2⁺ breast cancer cell line, HCC1806, when incubated with IMMU-132,had an increase in median fluorescence intensity (MFI) from 168(untreated baseline) to 546, indicating the increased presence of dsDNAbreaks, whereas the MFI for cells incubated with the non-bindingconjugate remained at baseline levels. In contrast, MFI for the Trop-2antigen-negative cell line, HCC1395, remained at baseline levelsfollowing treatment with either IMMU-132 or the non-binding controlconjugate. Thus, the specificity of IMMU-132 over an irrelevant ADC wasconclusively revealed by evidence of dsDNA breaks only inTrop-2-expressing cells incubated with the anti-Trop-2-bindingconjugate.

TABLE 9 Specificity of IMMU-132 anti-tumor activity in vitro using flowcytometry with phospho-H2AX (anti-histone)-stained cells.^(A) Medianfluorescence intensity HCC1806 HCC1395 Treatment (Trop-2⁺) (Trop-2⁻)Cell alone 4.25 5.54 Cell + anti-rH2AX-AF488 168 122 Cell + IMMU-132 +anti-rH2AX-AF488 546 123 Cell + hA20-SN38 + anti-rH2AX-AF488 167 123^(A)HCC1806 (Trop-2⁺) or HCC1395 (Trop-2⁻) were incubated at 4° C. withIMMU-132 or a non-binding control conjugate (anti-CD20-SN-38) for 30min, washed and incubated overnight at 37° C. in fresh drug-free media.Cells were harvested, fixed, and permeabilized, then stained with thefluorescently-conjugated anti-histone antibody (rH2AX-AF488) fordetection of double-stranded DNA breaks. The median fluorescenceintensity (MFI) is given for (a) background staining of the cells alone(no anti-histone antibody), (b) the background level of dsDNA breaks forthe cells that had no prior exposure to the conjugates, and (c) afterexposed to IMMU-132 or hA20-SN-38 conjugates.

In Vivo Efficacy of Sacituzumab Govitecan in TNBC Xenografts.

The efficacy of IMMU-132 was assessed in nude mice bearing MDA-MB-468TNBC tumors (not shown). IMMU-132 at a dose of 0.12 or 0.20 mg/kgSN-38-equivalents (0.15 and 0.25 mg IMMU-132/dose) induced significanttumor regression, compared to saline, irinotecan (10 mg/kg; ˜5.8 mg/kgSN-38 equivalents by weight), or a control anti-CD20 ADC,hA20-CL2A-SN-38, given at the same 2 dose levels (P<0.0017, area underthe curve, AUC). Since mice convert irinotecan to SN-38 more efficientlythan humans (38) (in our studies, it averaged ˜25%, see below), at thisirinotecan dose ˜145 to 174 μg of SN-38 would be produced, while theadministered dose of IMMU-132 contained only 9.6 μg. Nevertheless,because IMMU-132 selectively targeted SN-38 to the tumors, it was moreefficacious. These results corroborate findings in other solid tumormodels (Cardillo et al., 2011, Clin Cancer Res 17:3157-69) showing thatspecific targeting of a small amount of SN-38 to the tumor with IMMU-132is much more effective than a much larger dose of irinotecan, or forthat matter a mixture of hRS7 IgG with an equal amount of free SN-38(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The unconjugatedRS7 antibody, even at repeated doses of 1 mg per animal, did not showany antitumor effects (Cardillo et al., 2011, Clin Cancer Res17:3157-69). However, in-vitro studies with gynecological cancersexpressing Trop-2 have indicated cell killing with the RS7 mAb byantibody-dependent cellular cytotoxicity (Bignotti et al., 2010, Eur JCancer 46:944-53; Raji et al., 2011, J Exp Clin Cancer Res 30:106;Varughese et al., 2011, Gynecol Oncol 122:171-7; Varughese et al., 2011,Am J Obstet Gyneol 205:567). Also, a monovalent Fab of anotheranti-Trop-2 antibody has been reported to be therapeutically active invitro and in animal studies.

On therapy day 56, four of the seven tumors in mice given 0.12 mg/kg ofthe hA20-CL2A-SN-38 control ADC already had progressed to the endpointof 1.0 cm³ (not shown). At this time, these animals were treated withIMMU-132, electing to use the higher dose of 0.2 mg/kg in an attempt toaffect the progression of these much larger tumors. Despite thesubstantial size of the tumors in several animals, all mice demonstrateda therapeutic response, with tumors significantly smaller in size fiveweeks later (total volume [TV]=0.14±0.14 cm³ vs. 0.74±0.41 cm³,respectively; P=0.0031, two-tailed t-test). Similarly, we chose twoanimals in the irinotecan-treated group with tumors that progressed to˜0.7 cm³ and re-treated one with irinotecan and the other with IMMU-132(not shown). Within 2 weeks of ending treatment, the tumor in theirinotecan-treated animal decreased 23% and then began to progress,while the tumor treated with IMMU-132 had stabilized with a 60% decreasein tumor size. These results demonstrate that even in tumors thatcontinued to grow after exposure to SN-38 via a non-specific ADC, asignificantly enhanced therapeutic response could be achieved whentreated with the Trop-2-specific IMMU-132. However, specific therapeuticeffects with IMMU-132 were not achieved in MDA-MB-231 (not shown). Thiscell line had the lowest Trop-2 levels, but also was the least sensitiveto SN-38.

Mechanism of Action of IMMU-132 in TNBC—

The apoptotic pathway utilized by IMMU-132 was examined in the TNBC cellline, MDA-MB-468, and in the HER2⁺ SK-BR-3 cell line, in order toconfirm that the ADC functions on the basis of its incorporated SN-38(not shown). SN-38 alone and IMMU-132 mediated >2-fold up-regulation ofp21^(WAF1/Cip1) within 24 h in MDA-MB-468, and by 48 h, the amount ofp21^(WAF1/Cip1) in these cells began to decrease (31% and 43% with SN-38or IMMU-132, respectively). Interestingly, in the BERT′ SK-BR-3 tumorline, neither SN-38 nor IMMU-132 mediated the up-regulation ofp21^(WAF1/Cip1) above constitutive levels in the first 24 h, but as seenin MDA-MB-468 cells after 48-h exposure to SN-38 or IMMU-132, the amountof p21^(WAF1/Cip1) decreased >57%. Both SN-38 and IMMU-132 resulted incleavage of pro-caspase-3 into its active fragments within 24 h, butwith the greater degree of active fragments observed after exposure for48 h. Of note, in both cell lines, IMMU-132 mediated a greater degree ofpro-caspase-3 cleavage, with the highest level observed after 48 h whencompared to cells exposed to SN-38. Finally, SN-38 and IMMU-132 bothmediated poly ADP ribose polymerase (PARP) cleavage, starting at 24 h,with near complete cleavage after 48 h. Taken together, these resultsconfirm that IMMU-132 has a mechanism of action similar to that of freeSN-38 when administered in vitro.

Delivery of SN-38 by IMMU-132 vs. Irinotecan in a Human Tumor XenograftModel—

Constitutive products derived from irinotecan or IMMU-132 weredetermined in the serum and tumors of mice implanted s.c. with a humanpancreatic cancer xenograft (Capan-1) administered irinotecan (773 μg;SN-38 equivalents=448 μg) and IMMU-132 (1.0 mg; SN-38 equivalents=16μg).

Irinotecan cleared very rapidly from serum, with conversion to SN-38 andSN-38G seen within 5 min. None of the products was detected at 24 h. TheAUCs over a 6-h period were 21.0, 2.5, and 2.8 μg/mL·h for irinotecan,SN-38, and SN-38G, respectively (SN-38 conversion inmice=[2.5+2.8)/21=25.2%]). Animals given IMMU-132 had much lowerconcentrations of free SN-38 in the serum, but it was detected through48 h (not shown). Free SN-38G was detected only at 1 and 6 h, and was 3-to 7-times lower than free SN-38.

In the Capan-1 tumors excised from irinotecan-treated animals,irinotecan levels were high over 6 h, but undetectable a 24 h(AUC_(5min-6 h)=48.4 μg/g·h). SN-38 was much lower and detected onlythrough 2 h (i.e., AUC_(5min-2 h)=0.4 μg/g·h), with SN-38G values almost3-fold higher (AUC=1.1 μg/g·h) (not shown). Tumors taken from animalsgiven IMMU-132 did not have any detectable free SN-38 or SN-38G, butinstead, all SN-38 in the tumor was bound to IMMU-132. Importantly,since no SN-38G was detected in the tumors, this suggests SN-38 bound toIMMU-132 was not glucuronidated. The AUC for SN-38 bound to IMMU-132 inthese tumors was 54.3 μg/g·h, which is 135-fold higher than the amountof SN-38 in the tumors of animals treated with irinotecan over the 2-hperiod that SN-38 could be detected, even though mice given irinotecanreceived 28-fold more SN-38 equivalents than administered with IMMU-132(i.e., 448 vs 16 μg SN-38 equivalents, respectively)

Discussion

We describe a new ADC targeting Trop-2, and early clinical resultssuggest it is well tolerated and effective in patients with TNBC, aswell as other Trop-2⁺ cancers (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27). Due to its distinct properties, IMMU-132represents a second-generation ADC. Typically, ADCs require 4 broadattributes to be optimally-effective: (i) selective targeting/activity;(ii) binding, affinity, internalization, and immunogenicity of theantibody used in the ADC; (iii) the drug, its potency, metabolism andpharmacological disposition, and (iv) how the drug is bound to theantibody. Target selectivity is the most common requirement for allADCs, since this will play a major role in defining the therapeuticindex (ratio of toxicity to tumor vs. normal cells). Trop-2 appears tohave both a high prevalence on a number of epithelial cancers, but it isalso expressed by several normal tissues (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14; Trerotola et al., 2013, Oncogene 32:222-33;Stepan et al., 2011, 59:701-10), which could have impacted specificity.However, expression in normal tissues appears to be lower than incancers (Bignotti et al., 2010, Eur J Cancer 46:944-53), and Trop-2appears to be shielded by normal tissue architecture that limitsaccessibility to an antibody, whereas in cancer, these tissue barriersare compromised by the invading tumor. Evidence of this was apparentfrom initial toxicological studies in monkeys, where despite escalatingIMMU-132 doses to levels leading to irinotecan-like neutropenia anddiarrhea, histopathological damage to Trop-2-expressing normal tissuesdid not occur (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theseresults appear to have been confirmed clinically, where no specificorgan toxicity has been noted in patients to-date, except for the knowntoxicities of the parental compound, irinotecan (Bardia et al., 2014,San Antonio Breast Cancer Symposium, P5-19-27), which are moremanageable with IMMU-132.

A generally accepted and important criterion for ADC therapy is that theantibody should internalize, delivering its chemotherapeutic inside thecell, where it is usually metabolized in lysosomes. Despite IMMU-132'sinternalization, we believe that the linker in this ADC, which affordslocal release of SN-38 that likely can induce a bystander effect on thecancer cells, is another feature that sets this platform apart fromthose using an ultratoxic drug. Indeed, having an ultratoxic agentlinked stably to the IgG is the only configuration that would preserve auseful therapeutic window for those types of compounds. However, using amore moderately-toxic drug does not give the latitude to use a linkerthat would release the drug too early once in the circulation. Our groupexplored linkers that released SN-38 from the conjugate with differenthalf-lives in serum, ranging from ˜10 h to a highly stable linker, butit was the linker with the intermediate stability that provided the besttherapeutic response in mouse-human tumor xenograft models (Moon et al.,2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Chem Res15:6052-61). Since this initial work, we showed that a highly stablelinkage of SN-38 was significantly less effective than the CL2A linkerthat has a more intermediate stability in serum (Govindan et al., 2013,Mol Cancer Ther 12:968-78).

Another current tenet of ADC design is to use an ultra-cytotoxic drug tocompensate for low levels of antibody accretion in tumors, typically0.003 to 0.08% of the injected dose per gram (Sharkey et al., 1995,Cancer Res 55:5935s-45s). The current generation of ultratoxic-drugconjugates have found a drug:antibody substitutions of ≤4:1 to beoptimal, since higher ratios adversely affected their pharmacokineticsand diminished the therapeutic index by collateral toxicities (Hamblettet al., 2004, Clin Cancer Res 10:7063-70). In this second-generation ADCplatform, we elected to use an IgG-coupling method thatsite-specifically links the drug to the interchain disulfides throughmild reduction of the IgG, which exposes 8 binding sites. With theCL2A-SN-38 linker, we achieved a DAR of 7.6:1, with LC-MS data showingeach of the 8 coupling sites bears the CL2A linker, but apparently someSN-38 is lost during the manufacturing procedure. Nevertheless, 95% ofthe CL2A linker has 7-8 SN-38 molecules. We found subsequently that (a)coupling to these sites does not destabilize the antibody, and (b)conjugates prepared with these sites substituted at higher levels didnot compromise antibody binding, nor did it affect pharmacokineticproperties. Indeed, we demonstrated that conjugates prepared at themaximum substitution level had the best therapeutic response inmouse-human tumor xenograft models.

One of the more notable features of IMMU-132 from a tolerabilityperspective is that the SN-38 bound to IgG is not glucuronidated, whichis a critical step in the detoxification of irinotecan. With irinotecantherapy, most of the SN-38 generated is readily converted in the liverto the inactive SN-38G form. Estimates of the AUC for SN-38G show it isoften 4.5- to 32-times higher than SN-38 (Gupta et al., 1994, Cancer Res54:3723-25; Xie et al., 2002, J Clin Oncol 20:3293-301). SN-38G'ssecretion into the bile and subsequent deconjugation bybeta-glucuronidase produced by the intestinal flora is stronglyimplicated in the enterohepatic recirculation of SN-38 and the delayedsevere diarrhea observed with irinotecan (Stein et al., 2010, Ther AdvMed Oncol 2:51-63). After IMMU-132 administration, concentrations ofSN-38G were very low in our animal and clinical studies (e.g., in theserum of patients given IMU-132, only 20-40% of the free SN-38 levelsare in the form of SN-38G), providing strong evidence that SN-38 boundto IgG is largely protected from glucuronidation, even though the10-hydroxy position of the SN-38 is available. We speculate that lowlevels of SN-38G generated by IMMU-132 contributes to the lowerincidence and intensity of diarrhea in patients receiving this ADCcompared to irinotecan therapy.

Preventing glucuronidation of the SN-38 bound to the antibody may alsocontribute to improved therapeutic effects for SN-38 delivered to thetumor. Extracts of tumors from animals given irinotecan found highlevels of irinotecan, with 10-fold lower concentrations of SN-38 andSN-38G. In contrast, the only SN-38 found in the tumors of animals givenIMMU-132 was SN-38 bound to the IgG. We hypothesize that the conjugateretained in the tumor will eventually be internalized, thereby releasingits SN-38 payload, or SN-38 could be release outside the tumor cell;however, it would be released in its fully active form, with a lowerlikelihood of being converted to SN-38G, which occurs primarily in theliver. It is also important to emphasize that by coupling the linker tothe 20-hydroxy position of SN-38, the SN-38 is maintained in the activelactone form (Zhao et al., 2000, J Org Chem 65:4601-6). Collectively,these results suggest that IMMU-132 is able to deliver and concentrateSN-38 to Trop-2⁺ tumors in a selective manner compared to SN-38 derivedfrom non-targeted irinotecan, with the SN-38 delivered by IMMU-132likely being released in the tumor in the fully active,non-glucuronidated, lactone form.

Irinotecan is not conventionally used to treat breast cancer patients.However, the experiments shown here with TNBC cell lines indicate thatconcentrating higher amounts of SN-38 into the tumor enhances itsactivity. In both the MDA-MB-468 TNBC and HER2⁺ SK-BR-3 tumor lines,IMMU-132 mediated the activation of the intrinsic apoptotic pathway,with cleavage of pro-caspases into their active fragments and PARPcleavage. The demonstration of double-stranded DNA breaks of cancercells treated with IMMU-132 (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27) compared to an irrelevant SN-38 ADC)confirms the selective delivery of SN-38 into the target cells. Mostimportantly, these laboratory findings are confirmed by therapy ofpatients with heavily-pretreated, metastatic TNBC, where durableobjective responses have been observed (Bardia et al., 2014, San AntonioBreast Cancer Symposium, P5-19-27). It also appears that IMMU-132 isactive in patients with other cancers and who have failed a priortherapy regimen containing a topoisomerase I inhibitor (Starodub et al.,2015, Clin Cancer Res 21:3870-78).

In conclusion, the use of SN-38 conjugated at a very high ratio of drugto antibody, using a moderately-stable linker, is efficacious in animalmodels and also clinically, constituting a second-generation ADCplatform. Our findings indicate that Trop-2 is a clinically-relevant andnovel target in Trop-2+ solid tumors, particularly TNBC.

Example 17. Studies on the Mechanism of Action of IMMU-132

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CMS chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.

Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT=˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer xenograftsdemonstrate similar, significant anti-tumor effects in both models.Current Phase I/II clinical trials (ClinicalTrials.gov, NCT01631552)confirm anticancer activity of IMMU-132 in cancers expressing Trop-2,including gastric and pancreatic cancer patients.

Introduction

There will be an estimated 22,220 new cases of gastric cancer diagnosedin the United States this year, with a further 10,990 deaths attributedto this disease (Siegel et al., 2014, CA Cancer J Clin 64:9-29). While5-year survival rates are trending upward (currently at 29%), they arestill quite low when compared to most others, including cancers of thecolon, breast, and prostate (65%, 90%, and 100%, respectively). In fact,among human cancers, only esophageal, liver, lung, and pancreatic haveworse 5-year survival rates. Pancreatic cancer remains the fourthleading cause of all cancer deaths in the U.S., with a 5-year survivalrate of only 6% (Siegel et al., 2014, CA Cancer J Clin 64:9-29). It isclear from such grim statistics for gastric and pancreatic cancer thatnew therapeutic approaches are needed.

Trop-2 is a 45-kDa glycoprotein that belongs to the TACSTD gene family,specifically TACSTD22. Overexpression of this trans-membrane protein onmany different epithelial cancers has been linked to an overall poorprognosis. Trop-2 is essential for anchorage-independent cell growth andtumorigenesis (Wang et al., 2008, Mol Cancer Ther 7:280-85; Trerotola etal., 2013, Oncogene 32:222-33). It functions as a calcium signaltransducer that requires an intact cytoplasmic tail that isphosphorylated by protein kinase C12-14. Pro-growth signaling associatedwith Trop-2 includes NF-κB, cyclin D1 and ERK (Guerra et al., 2013,Oncogene 32:1594-1600; Cubas et al., 2010, Mol Cancer 9:253).

In pancreatic cancer, Trop-2 overexpression was observed in 55% ofpatients studied, with a positive correlation with metastasis, tumorgrade, and poor progression-free survival of patients who underwentsurgery with curative intent (Fong et al., 2008, Br J Cancer99:1290-95). Likewise, in gastric cancer 56% of patients exhibitedTrop-2 overexpression on their tumors, which again correlated withshorter disease-free survival and a poorer prognosis in those patientswith lymph node involvement of Trop-2-positive tumor cells (Muhlmann etal., 2009, J Clin Pathol 63:152-58). Given these characteristics and thefact that Trop-2 is linked to so many intractable cancers, Trop-2 is anattractive target for therapeutic intervention with an antibody-drugconjugate (ADC).

A general paradigm for using an antibody to target a drug to a tumorincludes several key features, among them: (a) an antigen target that ispreferentially expressed on the tumor versus normal tissue, (b) anantibody that has good affinity and is internalized by the tumor cell,and (c) an ultra-toxic drug that is coupled stably to the antibody(Panowski et al., 2014, mAbs 6:34-45). Along these lines, we developedan antibody, designated RS7-3G11 (RS7), that bound to Trop-2 in a numberof solid tumors (Stein et al., 1993, Int J Cancer 55:938-46; Basu etal., 1995, Int J Cancer 62:472-79) with nanomolar affinity (Cardillo etal., 2011 k, Clin Cancer Res 17:3157-69), and once bound to Trop-2, isinternalized by the cell (Shih et al., 1995, Cancer Res 55:5857s-63s).

By immunohistochemistry, Trop-2 is expressed in some normal tissues,though usually at much lower intensities when compared to neoplastictissue, and often is present in regions of the tissues with restrictedvascular access (Trerotola et al., 2013, Oncogene 32:222-33). Based onthese characteristics, RS7 was humanized and conjugated with the activemetabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Invitro cytotoxicity in numerous cell lines has found IC₅₀-values in thesingle digit nanomolar range for SN-38, compared to picomolar range formany of the ultra-toxic drugs currently used in ADCs (Cardillo et al.,2011, Clin Cancer Res 17:3157-69). While the prevailing opinion is touse ultra-toxic agents, such as auristatins or maytansines, to make ADCswith only 2-4 drugs per antibody linked stably to the antibody, suchagents have a narrow therapeutic window, resulting in renewed efforts tore-engineer ADCs to broaden their therapeutic index (Junutula et al.,2010, Clin Cancer Res 16:4769-78).

As one approach to diverge from this practice, we conjugated 7-8 SN-38molecules per antibody using a linker that releases SN-38 with half-lifeof ˜1 day in human serum. It is hypothesized that using a less stablelinker allows for SN-38 release at the tumor site after the ADC targetsthe cells, making the drug accessible to surrounding tumor cells and notjust cells directly targeted by the ADC. The resulting ADC,hRS7-CL2A-SN-38 (sacituzumab govitecan, or IMMU-132), has shownanti-tumor activity against a wide range of tumor types (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). More recently, IMMU-132 hasdemonstrated significant anti-tumor activity against a pre-clinicalmodel of triple negative breast cancer (TNBC) (Goldenberg et al., 2014,Poster presented at San Antonia Breast Cancer Symposium, December 9-13,Abstr. P5-19-08). Most importantly, in a current Phase I/II clinicaltrial, IMMU-132 has shown activity in TNBC patients (Bardia et al.,2014, Poster presented at San Antonia Breast Cancer Symposium, December9-13, Abstr. P5-19-2), thus validating this paradigm shift in ADCchemistry using a less toxic drug and a linker that releases SN-38 overtime rather than being totally dependent on internalization of the ADCto achieve activity.

SN-38 is a known topoisomerase-I inhibitor that induces significantdamage to a cell's DNA. It mediates the up-regulation of earlypro-apoptotic proteins, p53 and p21WAF1/Cip1, resulting in caspaseactivation and poly-ADP-ribose polymerase (PARP) cleavage. Expression ofp21WAF1/Cip1 is associated with G1 arrest of the cell cycle and is thusa hallmark of the intrinsic apoptotic pathway. We demonstratedpreviously that IMMU-132 likewise could mediate the up-regulation ofearly pro-apoptosis signaling events (p53 and p21WAF1/Cip1) resulting inPARP cleavage in NSCLC (Calu-3) and pancreatic (BxPC-3) cell linesconsistent with the intrinsic pro-apoptosis signaling pathway (Cardilloet al., 2011, Clin Cancer Res 17:3157-69).

Herein, we further characterize IMMU-132, with particular attentiontowards the treatment of solid cancers, especially human gastric andpancreatic tumors. Trop-2 surface expression across a range of solidtumor types is examined and correlated with in vivo expression in tumorxenografts. Mechanistic studies further elucidate the intrinsicpro-apoptotic signaling events mediated by IMMU-132, including evidenceof increased double stranded DNA (dsDNA) breaks and later caspaseactivation. Finally, clinically-relevant and non-toxic dosing schemesare compared in gastric and pancreatic carcinoma disease models, testingtwice-weekly, weekly, and every other week schedules to ascertain whichtreatment cycle may be best applied to a clinical setting without lossof efficacy.

Experimental Procedures

Cell Lines and Chemotherapeutics—

All human cancer cell lines used were purchased from the American TypeCulture Collection (ATCC) (Manassas, Va.). Each was maintained accordingto the recommendations of ATCC and routinely tested for mycoplasma, andall were authenticated by short tandem repeat (STR) assay by the ATCC.IMMU-132 (hRS7-SN-38) and control ADCs (anti-CD20 hA20-SN-38 andanti-CD22 hLL2-SN-38) were made as previously described and stored at−20° C. (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). SN-38 waspurchased (Biddle Sawyer Pharma, LLC, New York, N.Y.) and stored in 1 mMaliquots in DMSO at −20° C.

Trop-2 ELISA—

Recombinant human Trop-2 with a His-tag (Sino Biological, Inc., Bejing,China; Cat#10428-H09H) and recombinant mouse Trop-2 with a His-Tag (SinoBiological, Inc., Cat#50922-M08H) were plated onto Ni-NTA Hissorb strips(Qiagen GmbH Cat#35023) at 1 μg for 1 h at room temperature. The platewas washed four times with PBS-Tween (0.05%) wash buffer. Serialdilutions of hRS7 were made in 1% BSA-PBS dilution buffer to a testrange of 0.1 ng/mL to 10 μg/mL. The plates were then incubated for 2 hat room temperature before being washed four times followed by theaddition of a peroxidase conjugated secondary antibody (goat anti-human,Fc fragment specific; Jackson Immunoresearch Cat#109-036-098). After a45-min incubation, the plate was washed and a substrate solution(o-phenylenediamine dihydrochloride (OPD); Sigma, Cat# P828) added toall the wells. Plates were incubated in the dark for 15 min before thereaction was stopped with 4N sulfuric acid. The plates were read at 450nm on Biotek ELX808 plate reader. Data were analyzed and graphed usingPrism GraphPad Software (v4.03) (Advanced Graphics Software, Inc.;Encinitas, Calif.).

In Vitro Cell Binding—

LumiGLO Chemiluminescent Substrate System (KPL, Gaithersberg, Md.) wasused to detect antibody binding to cells. Briefly, cells were platedinto a 96 black-well, flat-clear-bottom plate overnight. Antibodies wereserially diluted 1:2 and added in triplicate, yielding a concentrationrange from 0.03 to 66.7 nM. After incubating for 1 h at 4° C., the mediawas removed and the cells washed with fresh cold media followed by theaddition of a 1:20,000 dilution of goat-antihuman horseradishperoxidase-conjugated secondary antibody (Jackson Immunoresearch, WestGrove, Pa.) for 1 h at 4° C. The plates were again washed before theaddition of the LumiGLO reagent. Plates were read for luminescence usingan Envision plate reader (Perkin Elmer, Boston Mass.). Data wereanalyzed by non-linear regression to determine the equilibriumdissociation constant (KD). Statistical comparisons of KD-values weremade with Prism GraphPad Software (v4.03) (Advanced Graphics Software,Inc.; Encinitas, Calif.) using an F-Test on the best-fit curves for thedata. Significance was set at P<0.05.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)—

A four-hour LDH-release assay was performed to evaluate ADCC activityelicited by IMMU-132, hRS7 IgG, hLL2-SN-38 and hLL2 IgG (hLL2 arenon-binding anti-CD22 conjugates for the solid tumor cell lines).Briefly, target cells (MDA-MB-468, NIH:OVCAR-3, or BxPC-3) were platedat 1×10⁴ cells/well in a 96-well, black, flat-bottom plate and incubatedovernight. The next day, peripheral blood mononuclear effector cells(PBMCs) were freshly isolated from a donor and added to assigned wellson the reaction plate at an E:T ratio of 50:1. Acquisition of humanPBMCs was done under the approval of the New England InstitutionalReview Board (Newton, Mass.). Test reagents were added to their assignedwells at a final concentration of 33.3 nM. One set of wells receivedADCC assay medium alone for background control and another set of wellsreceived cells alone plus TritonX100 for maximum cells lysis control.The plate was incubated for 4 h at 37° C. After 4 h, target cell lysiswas assessed by a homogenous fluorometric LDH release assay (CytoTox-One Homogenous Membrane Integrity Assay; Promega, Cat. G7891).

The plates were read (544 nm-590 nm) using an Envision plate reader(PerkinElmer LAS, Inc.; Shelton, Conn.). Data were analyzed by MicrosoftExcel. Percent specific lysis was calculated as follows:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Lysis}} = {\frac{{Experimental} - \left( {{Effector} + {{Target}\mspace{14mu} {Control}}} \right)}{{{Max}.\mspace{14mu} {Lysis}} - \left( {{Target}\mspace{14mu} {Control}} \right)} \times 100}$

where:

Experimental: effector+target cells+antibody

Effector+Target Control: effector+target cells

Max. Lysis: target cells+Triton-X100

Target Control: target cells only

Surface Plasmon Resonance Binding (BIACORE)—

Briefly, rhTrop-2/TACSTD2 (Sino Biological, Inc.) or recombinant humanneonatal receptor (FcRn), produced as described (Wang et al., 2011, DrugMetab Dispos 39:1469-77), was immobilized with an amine coupling kit (GEHealthcare; Cat. No. BR-1000-50) on a CM5 sensor chip (GE Healthcare;Cat. #BR-1000-12) following manufacturer's instructions for alow-density chip. Three separate sets of dilutions of hRS7 IgG andIMMU-132 were made in running buffer (400 nM, 200 nM, 100 nM, 50 nM and25 nM). Each set would make up a separate run on the BIACORE (BIACORE-X;Biacore Inc., Piscataway, N.J.) and data were analyzed usingBIAevaluation Software (Biacore Inc., v4.1). Analysis was performed witha 1:1 (Langmuir) Binding Model and Fit, using all five concentrationpoints for each sample run to determine the best fit (lowest χ² value).The KDvalue was calculated using the formula KD=kd1/ka1, where kd1 isthe dissociation rate-constant and ka1 is the association-rate constant.

Immunohistological Assessment of the Distribution of Trop-2 inFormalin-Fixed, Paraffin-Embedded Tissues—

Tumor xenografts were taken from mice, fixed in 10% buffered formalinand paraffin-embedded. After de-paraffination, 5 μm sections wereincubated with Tris/EDTA buffer (DaKo Target Retrieval Solution, pH 9.0;Dako, Denmark), at 95° C. for 30 min in a NxGen Decloaking Chamber(Biocare Medical, Concord, Calif.). Trop-2 was detected with a goatpolyclonal antihuman Trop-2 antibody at 10 μg/mL (R&D Systems,Minneapolis, Minn.) and stained with Vector VECTASTAINR ABC Kit (VectorLaboratories, Inc., Burlingame, Calif.). Normal goat antibody was usedas the negative control (R&D Systems, Minneapolis, Minn.). Tissues werecounterstained with hematoxylin for 6 seconds.

Trop-2 Surface Expression on Human Carcinoma Cell Lines—

Expression of Trop-2 on the cell surface is based on flow cytometry.Briefly, cells were harvested with Accutase Cell Detachment Solution(Becton Dickinson (BD), Franklin Lakes, N.J.; Cat. No. 561527) andassayed for Trop-2 expression using QuantiBRITE PE beads (BD Cat. No.340495) and a PE-conjugated anti-Trop-2 antibody (eBiosciences, Cat. No.12-6024) following the manufactures' instructions. Data were acquired ona FACSCalibur Flow Cytometer (BD) with CellQuest Pro software. Stainingwas analyzed with Flowjo software (Tree Star, Ashland Oreg.).

Pharmacokinetics—

Naive female NCr nude (nu/nu) mice, 8-10 weeks old, were purchased fromTaconic Farms (Germantown, N.Y.). Mice (N=5) were injected i.v. with 200μg of IMMU-132, parental hRS7, or modified hRS7-NEM (hRS7 treated withTCEP and conjugated with N-ethylmaleimide). Animals were bled viaretroorbital plexis at 30-min, 4-, 24-, 72- and 168-h post-injection.ELISA was utilized to determine serum concentrations of total hRS7 IgGby competing for the binding to an anti-hRS7 IgG idiotype antibody witha horseradish peroxidase conjugate of hRS7. Serum concentrations ofintact IMMU-132 were determined using an anti-SN-38 antibody to captureand a horseradish peroxidase-conjugated anti-hRS7 IgG antibody todetect. Pharmacokinetic (PK) parameters were computed bynoncompartmental analysis using Phoenix WinNonlin software (version 6.3;Pharsight Corp., Mountainview, Calif.).

Assessment of Double-Stranded DNA Breaks In Vitro—

For drug activity testing, BxPC-3 cells were seeded in 6-well plates at5×105 cells/well and held at 37° C. overnight. After 10 min cooling onice, cells were incubated with IMMU-132, hA20-SN-38, or hRS7-IgG at thefinal concentration of 20 μg/ml for 30 min on ice, washed three timeswith fresh media, and then returned to 37° C. to continue cultureovernight. The following morning, cells were trypsinized briefly, spundown, stained with Fixable Viability Stain 450 (BD Biosciences, SanJose, Calif.), washed with 1% BSA-PBS, and then fixed in 4% formalin for15 min, washed again and permeabilized in 0.15% Triton-X100 in PBS foranother 15 min. After washing twice with 1% BSA-PBS, cells wereincubated with mouse anti-γH2AX-AF488 (EMD Millipore Corporation,Temecula, Calif.) for 45 min at 4° C. The signal intensity of γH2AX wasmeasured by flow cytometry using a BD FACSCanto (BD Biosciences, SanJose, Calif.).

In Vivo Therapeutic Studies—

NCr female athymic nude (nu/nu) mice, 4-8 weeks old, were purchased fromTaconic Farms (Germantown, N.Y.). NCI-N87 gastric tumor xenografts wereestablished by harvesting cells from tissue culture and making a finalcell suspension 1:1 in matrigel (BD Bioscience; San Jose, Calif.), witheach mouse receiving a total of 1×10⁷ cells s.c. in the right flank. ForBxPC-3. xenografts of 1 g were harvested, and a tumor suspension made inHBSS to a concentration of 40% tumor w/v. This suspension was mixed 1:1with matrigel for a final tumor suspension of 20% w/v. Mice were theninjected with 300 μL s.c. Tumor volume (TV) was determined bymeasurements in two dimensions using calipers, with volumes defined as:L×w²/2, where L is the longest dimension of the tumor and w theshortest. For IHC, tumors were allowed to grow to approximately 0.5 cm³before the mice were euthanized and the tumors removed, formalin-fixedand paraffin-embedded. For therapy studies, mice were randomized intotreatment groups and therapy begun when tumor volumes were approximately0.25 cm³. Treatment regimens, dosages, and number of animals in eachexperiment are described in the Results and in the Figure legends. Thelyophilized IMMU-132 and control ADC (hA20-SN-38) were reconstituted anddiluted as required in sterile saline.

Mice were euthanized and deemed to have succumbed to disease once tumorsgrew to greater than 1.0 cm³ in size. Best responses to therapy weredefined as: partial response, shrinking >30% from starting size; stabledisease, tumor volumes shrinking up to 29% or increase no greater than20% of initial size; progression, tumors increase ≥20% either from theirstarting size or from their nadir. Time to progression (TTP) wasdetermined as time post-therapy initiation when the tumor grew more than20% in size from its nadir.

Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups prior to statistical analysis of growth curves.A two-tailed t-test was used to assess statistical significance betweenthe various treatment groups and controls, except for the salinecontrol, where a one-tailed t-test was used (significance at P≤0.05).Survival studies were analyzed using Kaplan-Meier plots (log-rankanalysis), using the Prism GraphPad Software (v4.03) software package(Advanced Graphics Software, Inc., Encinitas, Calif.).

Immunoblotting—

Cells (2×10⁶) were plated in 6-well plates overnight. The following daythey were treated with either free SN-38 (dissolved in DMSO) or IMMU-132at an SN-38 concentration equivalent to 0.4 μg/mL (1 μM). Parental hRS7was used as a control for the ADC. Cells were lysed in buffer containing10 mM Tris, pH 7.4, 150 mM NaCl, protease inhibitors and phosphataseinhibitors (2 mM Na2PO4, 10 mM NaF). A total of 20 μg protein wasresolved in a 4-20% SDS polyacrylamide gel, transferred onto anitrocellulose membrane and blocked by 5% non-fat milk in 1×TBS-T(Tris-buffered saline, 0.1% Tween-20) for 1 h at room temperature.Membranes were probed overnight at 4° C. with primary antibodiesfollowed by 1-h incubation with antirabbit secondary antibody (1:2500)at room temperature. Signal detection was done using a chemiluminescencekit (Supersignal West Dura, Thermo Scientific; Rockford, Ill.) with themembranes visualized on a Kodak Image Station 40000R. Primary antibodiesp21Waf1/Cip1 (Cat. No. 2947), Caspase-3 (Cat. No. 9665), Caspase-7 (Cat.No. 9492), Caspase-9 (Cat. No. 9502), PARP (Cat. No. 9542), (3-actin(Cat. No. 4967), pJNK1/2 (Cat. No. 4668), JNK (Cat. No. 9258), and goatanti-rabbit-HRP secondary antibody (Cat. No. 7074) were obtained fromCell Signal Technology (Danvers, Mass.).

Results

Trop-2 Expression Levels in Multiple Solid Tumor Cell Lines—

Surface expression of Trop-2 is evident in a variety of human solidtumor lines, including gastric, pancreatic, breast, colon, and lung(Table 10). There is no one tumor type that had higher expression aboveany other, with variability observed within a given tumor cell type. Forexample, within gastric adenocarcinomas, Trop-2 levels ranged from verylow 494±19 (Hs 746T) to high 246,857±64,651 (NCI-N87) surface moleculesper cell.

Gastrointestinal tumor xenografts stained for Trop-2 expression showedboth cytoplasmic and membrane staining (not shown). Staining intensitycorrelated well with the results for surface Trop-2 expressiondetermined by FACS analysis. For the pancreatic adenocarcinomas, allthree had homogenous staining, with BxPC-3 representing 2+ to 3+staining. NCI-N87 gastric adenocarcinoma had a more heterogeneousstaining pattern, with 3+ staining of the apical lining of the glandsand less pronounced staining of surrounding tumor cells. COLO 205demonstrated only very focal 1+ to 2+ staining, whereas HT-29 showedvery rare 1+ staining of a few cells.

TABLE 10 Trop-2 surface expression levels in various solid tumor linesvia FACS analysis. a Number of Surface Trop-2 Molecules per Cell CellLine Mean ± SD Gastric NCI-N87 246,857 ± 64,651 AGS  53,756 ± 23,527 Hs746T 494 ± 19 Pancreatic BxPC-3 493,773 ± 97,779 CFPAC-1 162,871 ±28,161 Capan-1 157,376 ± 36,976 HPAF-II 115,533 ± 28,627 Breast (TN)MDA-MB-468 301,603 ± 29,470 HCC38 181,488 ± 69,351 HCC1806  91,403 ±20,817 MDA-MB-231 32,380 ± 5,460 Breast SK-BR-3 (HER2+) 328,281 ± 47,996MCF-7 (ER2+) 110,646 ± 17,233 Colon COLO 205 58,179 ± 6,909 HT-29  68 ±17 NSCLC Calu-3 128,201 ± 50,708 Sq. Cell Lung SK-MES-1 29,488 ± 5,824Acute T-Cell Leukemia Jurkat 0 a Three separate assays were performed,with the mean and standard deviation provided.

IMMU-132 Binding Characteristics—

To further demonstrate that hRS7 does not cross-react with murineTrop-2, an ELISA was performed on plates coated with either recombinantmurine Trop-2 or human Trop-2 (not shown). Humanized RS7 specificallybound only to the human Trop-2 (KD=0.3 nM); there was nocross-reactivity with the murine Trop-2. Control polyclonal rabbitanti-murine Trop-2 and antihuman Trop-2 antibodies did cross-react andbound to both forms of Trop-2 (data not shown).

IMMU-132 binding to multiple cell lines was examined, with comparison toparental hRS7 as well as to modified hRS7, hRS7-NEM (hRS7 treated withTCEP and conjugated with N-ethylmaleimide) (not shown). In all cases,calculated KD-values were in the sub-nanomolar range, with nosignificant differences between hRS7, IMMU-132, and hRS7-NEM within agiven cell line.

Comparisons in binding of IMMU-132 and hRS7 were further investigatedusing surface plasmon resonance (BIACORE) analysis (not shown). Alow-density Trop-2 biosensor chip (density=1110 RU) was utilized withrecombinant human Trop-2. Not only did three independent binding runsdemonstrate that IMMU-132 is not affected adversely by theSN-38-conjugation process, but it demonstrated a higher binding affinityto Trop-2 than hRS7 (0.26±0.14 nM vs. 0.51±0.04 nM, respectively;P=0.0398).

Mechanism of Action: ADCC and Intrinsic Apoptosis Signaling Pathways—

ADCC activity of IMMU-132 was compared to hRS7 in three different celllines, TNBC (MDAMB-468), ovarian (NIH:OVCAR-3), and pancreatic (BxPC-3)(not shown). In all three, hRS7 significantly mediated cell lysiscompared to all other treatments, including IMMU-132 (P<0.0054). ADCCdecreased by more than 60% when IMMU-132 was used to target the cells ascompared to hRS7. For example, in MDA-MB-468, specific lysis mediated byhRS7 was 29.8±2.6% versus 8.6±2.6% for IMMU-132 (not shown; P<0.0001).Similar loss in ADCC activity was likewise observed in NIH:OVCAR-3 andBxPC-3 (not shown; P<0.0001 and P<0.0054; respectively). This diminishedADCC activity appears to be the result of changes to the antibody duringthe conjugation process, since this same loss in specific cell lysis wasevident with hRS7-NEM, which lacks the CL2A-SN-38 linker, having thecysteines blocked instead with N-ethylmaleimide (not shown). There is noCDC activity associated with hRS7 or IMMU-132 (data not shown).

IMMU-132 has been shown previously to mediate the up-regulation of earlypro-apoptosis signaling events (p53 and p21WAF1/Cip1), ultimatelyleading to the cleavage of PARP20. In order to better define theapoptotic pathway utilized by IMMU-132, the NCI-N87 human gastriccarcinoma and BxPC-3 pancreatic adenocarcinoma cell lines were exposedto 1 μM of free SN-38 or the equivalent amount of IMMU-132 (not shown).Both free SN-38 and IMMU-132 mediate the up-regulation of p21WAF1/Cip1,though it is not until 48 h that the up-regulation between the NCI-N87cells exposed to free SN-38 versus IMMU-132 are the same (not shown),whereas in BxPC-3 maximum up-regulation is evident within 24 h (notshown). Both free SN-38 and IMMU-132 demonstrate cleavage ofpro-caspase-9 and -7 within 48 h of exposure. Procaspase-3 is cleaved inboth cell lines with the highest degree of cleavage observed after 48 h.Finally, both free SN-38 and IMMU-132 mediated PARP cleavage. This firstbecomes evident at 24 h, with increased cleavage at 48 h. Takentogether, these data confirm that the SN-38 contained in IMMU-132 hasthe same activity as free SN-38.

In addition to these later apoptosis signaling events, an earlier eventassociated with this pathway, namely the phosphorylation of JNK (pJNK),is also evident in BxPC-3 cells exposed for a short time to either freeSN-38 or IMMU-132, but not naked hRS7 (not shown). Increased amounts ofpJNK are evident by 4 h, with no appreciable change at 6 h. There is ahigher intensity of phosphorylation in the cells exposed to free-SN-38as compared to IMMU-132, but both are substantially higher thancontrols. As an end-point for the mechanism of action of IMMU-132,measurements of dsDNA breaks were made in BxPC-3 cells. Exposure ofBxPC-3 to IMMU-132 for only 30 min resulted in a greater than two-foldinduction of γH2AX when compared to a non-targeting control ADC (Table11). Approximately 70% of the cells were positive for γH2AX stainingversus <20% for naked hRS7, hA20-SN-38 irrelevant ADC, and untreatedcontrols (P<0.0002).

TABLE 11 IMMU-132-mediated dsDNA breaks in BxPC-3: γH2AX induction.^(a)Treatment Mean Fluorescence Intensity Percent Positive Untreated 2516 ±191 18.8 ± 6.3 hRS7 2297 ± 18  13.0 ± 0.6 hA20-SN-38 2246 ± 58  12.7 ±2.4 IMMU-132 5349 ± 234 69.0 ± 4.1 ^(a)IMMU-132 vs. all 3 controltreatments, P < 0.0002 (onetailed t-Test; N = 3).

Pharmacokinetics of IMMU-132—

Binding to the human neonatal receptor (FcRn) was determined by BIACOREanalysis (not shown). Using a low-density FcRn biosensor chip(density=1302 RU), three independent binding runs at five differentconcentrations (400 to 25 nM) were conducted for each agent. Overall,both hRS7 and IMMU-132 demonstrate KD-values in the nanomolar range(92.4±5.7 nM and 191.9±47.6 nM, respectively), with no significantdifference between the two. Mice were injected with IMMU-132, with theclearance of IMMU-132 versus the hRS7 IgG compared to the parental hRS7using two ELISAs (not shown). Mice injected with hRS7 demonstrated abiphasic clearance pattern (not shown) that was similar to what wasobserved for the hRS7 targeting portion of IMMU-132 (not shown), withalpha and beta half-lives of approximately 3 and 200 h, respectively. Incontrast, a rapid clearance of intact IMMU-132 was observed with ahalf-life of 11 h and mean residence time (MRT) of 15.4 h (not shown).

To further confirm that disruption of interchain disulfide bonds doesnot alter the PK of the targeting antibody, the PK of parental hRS7 wascompared to modified hRS7 (hRS7-NEM). There were no significantdifferences noted between either agent in terms of half-life, Cmax, AUC,clearance, or MRT (not shown).

IMMU-132 Efficacy in Human Gastric Carcinoma Xenografts—

Efficacy of IMMU-132 has been demonstrated previously in non-small-celllung, colon, TNBC, and pancreatic carcinoma xenograft models (Cardilloet al., 2011, Clin Cancer Res 17:3157-69; Goldenberg et al., Posterpresented at San Antonio Breast Cancer Symposium, December 9-13, AbstrP5-19-08). To further extend these findings to other gastrointestinalcancers, IMMU-132 was tested in mice bearing a human gastric carcinomaxenograft, NCI-N87 (not shown). Treatment with IMMU-132 achievedsignificant tumor regressions compared to saline and non-targeting hA20(anti-CD20)-SN-38 ADC controls (not shown; P<0.001). There were 6 of 7mice in the IMMU-132 group that were partial responders that lasted formore than 18 days after the last therapy dose was administered to theanimals. This resulted in a mean time to progression (TTP) of 41.7±4.2days compared to no responders in the control ADC group, with a TTP of4.1±2.0 days (P<0.0001). Overall, the median survival time (MST) forIMMU-132-treated mice was 66 days versus 24 days for control ADC and 14days for saline control animals (not shown; P<0.0001).

Clinically-Relevant Dosing Schemes—

The highest repeated doses tolerated of IMMU-132 currently being testedclinically are 8 and 10 mg/kg given on days 1 and 8 of 21-day cycles. Ahuman dose of 8 mg/kg translates to a murine dose of 98.4 mg/kg, orapproximately 2 mg to a 20 g mouse. Three different dose schedules offractionated 2 mg of IMMU-132 were examined in a human pancreaticadenocarcinoma xenograft model (BxPC-3). This total dose wasfractionated using one of three different dosing schedules: one groupreceived two IMMU-132 doses of 1 mg (therapy days 1 and 15), onereceived four doses of 0.5 mg (therapy days 1, 8, 22, and 29), and thefinal group eight doses of 0.25 mg (therapy days 1, 4, 8, 11, 22, 25,29, and 32). All three dosing schemes provided a significant anti-tumoreffect when compared to untreated control animals, both in terms oftumor growth inhibition and overall survival (not shown; P<0.0009 andP<0.0001, respectively). There are no significant differences in TTPbetween the three different treatment groups, which ranged from22.4±10.1 days for the 1-mg dosing group to 31.7±14.5 days for the0.25-mg dosing group (TTP for untreated control group=5.0±2.3 days).

A similar dose schedule experiment was performed in mice bearing NCI-N87human gastric tumor xenografts (not shown). All three dose schedules hada significant anti-tumor effect when compared to untreated control micebut were no different from each other (AUC; P<0.0001). Likewise, interms of overall survival, while all three dose schedules provided asignificant survival benefit when compared to untreated control(P<0.0001), there were no differences between any of these threedifferent schedules.

To further discriminate possible dosing schemes, mice bearing NCI-N87tumors were subjected to chronic IMMU-132 dosing in which mice received0.5 mg injections of IMMU-132 once a week for two weeks followed by oneweek off before starting another cycle (not shown), as in the currentclinical trial schedule. In all, four treatment cycles were administeredto the animals.

This dosing schedule slowed tumor growth with a TTP of 15.7±11.1 daysversus 4.7±2.2 days for control ADC-treated mice (P=0.0122). Overall,chronic dosing increased the median 19 survival 3-fold from 21 days forcontrol ADC-treated mice to 63 days for those animals administeredIMMU-132 (P=0.0001). Importantly, in all these different dosing schemeevaluations, no treatment-related toxicities were observed in the miceas demonstrated by no significant loss in body weight (data not shown).

Discussion

In a current Phase I/II clinical trial (ClinicalTrials.gov,NCT01631552), IMMU-132 (sacituzumab govitecan) is demonstratingobjective responses in patients presenting with a wide range of solidtumors (Starodub et al., 2015, Clin Cancer Res 21:3870-78). As thisPhase I/II clinical trial continues, efficacy of IMMU-132 needs to befurther explored in an expanding list of Trop-2-positive cancers.Additionally, the uniqueness of IMMU-132, in contrast to otherclinically relevant ADCs that make use of ultratoxic drugs, needs to befurther elucidated as we move forward in its clinical development.

The work presented here further characterizes IMMU-132 and demonstratesits efficacy against gastric and pancreatic adenocarcinoma atclinically-relevant dosing schemes. The prevailing view of a successfulADC is that it should use an antibody recognizing an antigen with hightumor expression levels relative to normal tissue and one thatpreferably internalizes when bound to the tumor cells (Panowski et al.,2014, mAbs 6:34-45). All of the currently approved ADCs have used anultra-toxic drug (pM IC50) coupled to the antibody by a highly stablelinker at low substitution ratios (2-4 drugs per antibody). IMMU-132diverges from this paradigm in three main aspects: (i) SN-38, amoderately cytotoxic drug (nM IC₅₀), is used as the chemotherapeuticagent, (ii) SN-38 is conjugated site-specifically to 8 interchain thiolsof the antibody, yielding a substitution of 7.6 drugs per antibody, and(iii) a carbonate linker is used that is cleavable at low pH, but willalso release the drug with a half-life in serum of ˜24 h (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). IMMU-132 is composed of anantibody that internalizes after binding to an epitope, as we haveshown, that is specific for human Trop-2, which is highly expressed onmany different types of epithelial tumors, as well as at lowerconcentrations in their corresponding normal tissues (Shih et al., 1995,Cancer Res 55:5857s-63s). Despite the presence in normal tissues, priorstudies in monkeys, which also express Trop-2 in similar tissues,indicated relatively mild and reversible histopathological changes evenat very high doses where dose-limiting neutropenia and diarrheaoccurred, suggesting the antigen in the normal tissues was sequesteredin some manner, or that the use of a less toxic drug spared these normaltissues from severe damage (Cardillo et al., 2011, Clin Cancer Res17:3157-69).

Herein, we expanded an assessment of Trop-2 expression on multiple humansolid tumor lines, examining in vitro expression in a more quantitativemanner than reported previously, but also, importantly, in xenograftsthat illustrate Trop-2 expression ranging from homogenous (e.g., NCIN87)to very focal (e.g., COLO 205). Overall, surface expression levels ofTrop-2 determined in vitro correlated with staining intensity upon IHCanalysis of xenografts. It is particularly interesting that even in atumor like COLO 205, where there are only focal pockets ofTrop-2-expressing cells revealed by immunohistology, IMMU-132 was stillcapable of eliciting specific tumor regressions, suggesting that abystander effect may occur as a result of the release of SN-38 from theconjugate bound to the antigen-presenting cells (Cardillo et al., 2011,Clin Cancer Res 17:3157-69). Indeed, SN-38 readily penetrates cellmembranes, and therefore its local release within the tumormicroenvironment provides another mechanism for its entry into cellswithout requiring internalization of the intact conjugate. Importantly,the SN-38 bound to the conjugate remains in a fully active state;namely, it is not glucuronidated and would be in the lactone ring format the time of release (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). This property is unique, distinguishing IMMU-132's abilityto localize a fully active form of SN-38 in a more selective manner thanany of the other slow-release SN-38 or irinotecan agents studied todate.

The Phase I clinical trial with IMMU-132 identified 8 to 10 mg/kg givenweekly for two weeks on a 21-day cycle for further investigation inPhase II (Starodub et al., 2015, Clin Cancer Res 21:3870-78). Patientswith a wide range of metastatic solid tumors, including pancreatic andgastric cancers, have shown extended periods of disease stabilizationafter relapsing to multiple prior therapies (Starodub et al., 2015, ClinCancer Res 21:3870-78; Starodub et al., 2014, J Clin Oncol 32:5s (SupplAbstr 3032)). Additional studies in xenograft models were undertaken todetermine if different dosing schedules may be more efficacious. To thisend, the equivalent to the human dose of 8 mg/kg (mouse dose of 98.4mg/kg) was fractionated over three different dosing schedules, includingevery other week, weekly, or twice-weekly on a 21-day cycle. In thepancreatic and gastric tumor models, no significant difference intherapeutic responses were observed for all three schedules, with tumorsprogressing only after therapy was discontinued. Therefore, these datasupport the continued use of the once-weekly dosing regimen currentlybeing pursued clinically.

With clinical trials recommending an IMMU-132 each treatment dose of 8to 10 mg/kg (Starodub et al., 2015, Clin Cancer Res 21:3870-78), it wasimportant to examine whether the antibody alone might contribute to theIMMU-132's activity. Previous studies in nude mice-human xenograftmodels had included unconjugated hRS7 IgG alone (e.g., repeated doses of25 to 50 mg/kg), with no evidence of therapeutic activity (Cardillo etal., 2011, Clin Cancer Res 17:3157-69); however, studies in mice cannotalways predict immunological functionality. ADCC activity of hRS7 invitro has been reported in Trop-2-positive ovarian and uterinecarcinomas (Raji et al., 2011, J Exp Clin Cancer Res 30:106; Bignotti etal., 2011, Int J Gynecol Cancer 21:1613-21; Varughese et al., 2011, Am JObstet Gynecol 205:567; Varughese et al., 2011, Cancer 117:3163-72). Weconfirmed unconjugated hRS7 ADCC activity in three different cellslines, but found IMMU-132 lost 60-70% of its effector function. Sincethe reduced/NEM-blocked IgG has a similar loss of ADCC activity, itappears that the attachment of the CL2A-SN-38 component was not, initself, responsible.

Antibodies also can elicit cell death by acting on various apoptoticsignaling pathways. However, we did not observe any effects of theunconjugated antibody in a number of apoptotic signaling pathways, butinstead noted IMMU-132 elicited similar intrinsic apoptotic events asSN-38. Early events include the phosphorylation of JNK1/2 as well as theup-regulation of p21WAF1/Cip1 leading to the activation of caspase-9,-7, and -3, with the end result of PARP cleavage and significant levelsof dsDNA breaks, as measured by increased amounts of phosphorylatedhistone H2AX (γH2AX)41. These data suggest that IMMU-132's primarymechanism of action is related to SN-38.

Surface plasmon resonance (BIACORE) analysis did not detect asignificant difference in IMMU-132's binding to the human neonatalreceptor (FcRn), despite the average binding levels being ˜2-fold lowerfor IMMU-132. FcRn binding has been linked to an extended IgG half-lifein serum (Junghans & Anderson, 1996, Proc Natl Acad Sci USA 93:5512-16),but because an antibody's affinity for FcRn in vitro may not correlatewith in vivo clearance rates (Datta-Mannan et al., 2007, J Biol Chem282:1709-17), the overall importance of this finding is unknown.Previous experiments in tumor-bearing mice using ¹¹¹In-DTPA-IMMU-132revealed that the conjugate cleared at a somewhat faster rate from theserum than ¹¹¹In-DTPA-hRS7, although both had similar tumor uptake(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). In the currentstudies, an ELISA assay that also measured the clearance of the IgGcomponent found IMMU-132 and the reduced and NEM-blocked IgG cleared atsimilar rates as unconjugated hRS7, suggesting that the coupling to theinterchain disulfides does not destabilize the antibody. As expected,when using an ELISA that monitored the clearance of the intact conjugate(capturing using an anti-SN-38 antibody and probe with an anti-idiotypeantibody), its clearance rate was faster than when monitoring only theIgG component. This difference simply reflects SN-38's release from theconjugate with a half-life of ˜1 day. We also have examined theclearance rates of sacituzumab govitecan conjugates prepared atdifferent substitution levels by ELISA, and again found no appreciatedifference in their clearance rates (Goldenberg et al., 2015, Oncotarget8:22496-512). Overall, these data suggest that mild reduction of theantibody, with the subsequent site-specific modification of some or allinterchain disulfides, has minimal if any impact on the serum clearanceof the IgG, but IMMU-132's overall clearance rate will be definedlargely by the rate of release of SN-38 from the linker.

Additionally, extensive cell-binding experiments demonstrated nosignificant difference in the binding of IMMU-132, the unconjugatedantibody, or the NEM-modified antibody, suggesting that thesite-specific linkage to the interchain disulfides protects theantigen-binding properties of the antibody. Interestingly, when analyzedby BIACORE, which more accurately measures the on-rate and off-rate inaddition to overall affinity, IMMU-132 had a significant 2-foldimprovement in calculated KD-values for Trop-2 binding when compared tonaked hRS7.

We speculate that this improvement may be result of the addedhydrophobicity when SN-38 is conjugated to the antibody. Hydrophobicresidues, as well as hydrophobicity of enclosed regions of proteinbinding sites, have been shown to impart a stronger affinity for theepitope (Park et al., 2000, Nat Biotechnol 18:194-98; Berezov et al.,2001, J Med Chem 44:2565-74; Young et al., 2007, Proc Natl Acad Sci USA104:808013). These regions do not have to be at the protein-proteininterface, but can lie in surrounding, less energetically contactresidues (Li et al., 2005, Structure 13:297-307). While none of theSN-38 conjugation sites are present in the complement-determiningregions (CDR) of hRS7, the prospects that the SN-38 on the antibody maydisplace some of the water molecules around the epitope, resulting inthe improved binding affinity observed for IMMU-132 relative to nakedhRS7, cannot be discounted.

Most efforts in ADC development have been directed towards using astable linker and an ultratoxic drug, with preclinical studiesindicating the specific optimal requirements for those conjugates(Panowski et al., 2014, mAbs 6:34-45; Phillips et al., 2008, Cancer Res68:9280-90). For example, a comparison of T-DM1 to another less stablederivative, T-SSPDM1, revealed that intact T-SSP-DM1 cleared at anapproximately 2-fold faster rate than T-DM-1 in non-tumor-bearing mice(Phillips et al., 2008, Cancer Res 68:9280-90; Erickson et al., 2012,Mol Cancer Ther 11:1133-42), with 1.5-fold higher levels of T-DM1compared to T-SSPDM1 in the tumors. Unexpectedly, and most interesting,was the finding that the amount of free, active maytansinoid catabolitesin the targeted tumors was very similar between the two ADCs (Ericksonet al., 2012, Mol Cancer Ther 11:1133-42).

In other words, T-SSP-DM1 was able to overcome its deficiencies inlinker stability due to the fact that the lower stability resulted inmore efficient release of the drug at the tumor than the more stableT-DM1. Not surprisingly, this equivalency of active drug-catabolitebetween the two ADCs in the tumors resulted in similar anti-tumoreffects in tumor-bearing animals. Ultimately, T-DM1 was chosen based ontoxicity issues that arise when using an ultra-toxic drug and lessstable linkers (Phillips et al., 2008, Cancer Res 68:9280-90). SinceSN-38 is at least a log-fold less toxic than these maytansines, itsrelease from the ADC is expected to have less toxicity. However, evenwith its release in serum, the amount of SN-38 localized in humangastric or pancreatic tumor xenografts was up to 136-fold higher than intumor-bearing mice injected with irinotecan doses that had >20-foldhigher SN-38 equivalents (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). While we have tested more stable linkers in the developmentof IMMU-132, they were significantly less effective in xenograft tumormodels than IMMU-132 (Govindan et al., 2013, Mol Cancer Ther 12:968-78).

Similarly, linkers that released SN-38 more quickly (e.g., serumhalf-life of ˜10 h) also were less effective in xenograft models (Moonet al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin ChemRes 15:6052-61), suggesting that there is an optimal window at which therelease of SN-38 leads to improved efficacy. Thus, current datademonstrate that IMMU-132 is a more efficient way to target and releasethe drug at the tumor than irinotecan.

Early clinical studies have shown encouraging objective responses invarious solid tumors, and importantly have indicated a better safetyprofile, with a lower incidence of diarrhea, than irinotecan therapy(Starodub et al., 2015, Clin Cancer Res 21:3870-78).

In summary, IMMU-132 (sacituzumab govitecan) is a paradigm-shift in ADCdevelopment. It uses a moderately-stable linker to conjugate 7-8molecules of the more tolerable active metabolite of irinotecan, SN-38,to an anti-Trop-2 antibody. Despite these seemingly counterintuitivecharacteristics vis-a-vis ultra-toxic ADCs, non-clinical studies havedemonstrated that IMMU-132 very effectively targets Trop-2-expressingtumors with significant efficacy and no appreciable toxicity. In earlyphase I/II clinical trials against a wide range of solid tumors,including pancreatic, gastric, TNBC, small-cell and non-small-cell lungcarcinomas, IMMU-132 is likewise exhibiting anti-tumor effects withmanageable toxicities in these patients, with no immune responses toeither the IgG or SN-38 detected, even after many months of dosing(Starodub et al., 2015, Clin Cancer Res 21:3870-78). Given the elevatedexpression of Trop-2 on such a wide variety of solid tumors, IMMU-132continues to be studied clinically, especially in advanced cancers thathave been refractory to most current therapy strategies.

Example 18. Further Results from Phase VII Clinical Studies

Triple-Negative Breast Cancer (TNBC)

The phase I/II clinical trial (NCT01631552) discussed in the Examplesabove has continued, accruing 56 TNBC patients who were treated with 10mg/kg. The patient population had previously been extensively treatedbefore initiating IMMU-132 therapy, with at least 2 prior lines oftherapy including taxane treatment. Previous treatments includedcyclophosphamide, doxorubicin, carboplatin, gemcitabine, capecitabine,eribulin, cisplatinum, anastrozole, vinorelbine, bevacizumab andtamoxifen. Despite this extensive treatment history TNBC patientsresponded well to IMMU-132, with 2 confirmed complete responses (CR), 13partial responses (PR) and 25 stable disease (SD), for an objectiveresponse rate of 29% (15/52) (not shown). Adding the incidence of CRplus PR plus SD, treatment in TNBC resulted in a 71% favorable responserate for IMMU-132 treated patients (not shown). The median time toprogression in this heavily pretreated population of TNBC patients was9.4 months, with a range of 2.9 to 14.2 months to date. However, 72% ofpatients in the study were still ongoing treatment.

Metastatic NSCLC

The clinical trial is also ongoing for patients with metastaticnon-small cell lung cancer (NSCLC), with 29 assessable patients accruedto date, who were treated with 8 or 10 mg/kg IMMU-132. The bestresponses by RESIST 1.1 criteria were determined (not shown). Out of 29patients, there were 8 PR and 13 SD. The time to progression for NSCLCpatients showed that 21/33 (64%) of NSCLC patients exhibited PR or SD(not shown). The median time to progression was 9/4 months, with a rangefrom 1.8 to 15.5+ months and 47% of patients still undergoing treatment.Progression-free survival in NSCLC patients treated with 8 or 10 mg/kgIMMU-132 was determined (not shown). Median PFS was 3.4 months at 8mg/kg and 3.8 months at 10 mg/kg. However, studies are still ongoing andthe median progression-free survival numbers are likely to improve.

Metastatic SCLC

Comparable results in metastatic SCLC patients were observed (notshown). Time to progression (not shown) showed a median of 4.9 months,with a range of 1.8 to 15.7+ months and 7 patients still undergoingtreatment with IMMU-132. The progression free survival (not shown)showed a median PFS of 2.0 months at 8 mg/kg and 3.6 months at 10 mg/kg.The median OS was 8.1 months at 8 mg/kg and could not be determined yetfor 10 mg/kg.

Urothelial Cancer

Similar results were obtained with urothelial cancer patients treatedwith 8 or 10 mg/kg IMMU-132. The best response data for 11 assessablepatients showed 6 PR and 2 SD (not shown). Time to progression (notshown) showed a median of 8.1 months, with a range of 3.6 yo 9.7+months.

In summary, the continuing phase I/II clinical trial shows superiorefficacy of IMMU-132, when administered at the recited dosages of ADC,in at least TNBC, NSCLC, SCLC and urothelial cancers. The superiortherapeutic effect in these heavily pretreated and resistant metastaticcancers occurred without inducing severe toxicities that might precludeclinical use. IMMU-132 showed an acceptable safety profile in heavilypretreated patients with diverse solid cancers, and a median of 2-5prior therapies. Only neutropenia showed an incidence of greater than20% of the patient population for Grade 3 or higher adverse reactions.The study further demonstrates that repeated doses of IMMU-132 may beadministered to human patients, at therapeutic dosages, without evokinginterfering host anti-IMMU-132 antibodies. These results demonstrate thesafety and utility of IMMU-132 for treating diverse Trop-2 positivecancers in human patients.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating a Trop-2 positive cancer comprisingadministering to a human patient with a Trop-2 positive cancer an ADCcomprising SN-38 conjugated to a linker moiety attached to ananti-TROP-2 antibody or antigen-binding fragment thereof, wherein thepatient has relapsed from or is refractory to treatment with acheckpoint inhibitor, wherein the checkpoint inhibitor is an antibodythat binds to PD-1 or PD-L1.
 2. The method of claim 1, wherein thecheckpoint inhibitor is selected from the group consisting of MPDL3280A,pembrolizumab, nivolumab, pidilizumab, MDX-1105, durvalumab, BMS-936559,and tremelimumab.
 3. The method of claim 1, wherein the checkpointinhibitor is MPDL3280A.
 4. The method of claim 1, wherein theanti-Trop-2 antibody is an hRS7 antibody comprising the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2);and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO:6).
 5. The method of claim 1, wherein theanti-Trop-2 antibody competes for binding to Trop-2 with an antibodycomprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ IDNO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3)and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).6. The method of claim 1, wherein the linker is CL2A and the structureof the ADC is MAb-CL2A-SN-38

MAb-CL2A-SN-38.
 7. The method of claim 6, wherein the anti-Trop-2antibody is an hRS7 antibody comprising the light chain CDR sequencesCDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (NYGMN,SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO:6).
 8. The method of claim 1, wherein thecancer is selected from the group consisting of colorectal, lung,stomach, urinary bladder, renal, pancreatic, breast, ovarian, uterine,esophageal and prostatic cancer.
 9. The method of claim 1, wherein thecancer is triple negative breast cancer.
 10. The method of claim 1,wherein the cancer is selected from the group consisting oftriple-negative breast cancer, HER+, ER+, progesterone+ breast cancer,metastatic non-small-cell lung cancer, metastatic small-cell lungcancer, metastatic endometrial cancer, metastatic urothelial cancer andmetastatic pancreatic cancer.
 11. The method of claim 1, wherein the ADCis administered at a dosage of between 3 mg/kg and 18 mg/kg.
 12. Themethod of claim 11, wherein the dosage is selected from the groupconsisting of 3 mg/kg, 4 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10mg/kg, 11 mg/kg, 12 mg/kg, 16 mg/kg and 18 mg/kg.
 13. The method ofclaim 1, wherein the ADC is administered at a dosage of between 8 mg/kgand 12 mg/kg.
 14. The method of claim 1, wherein the ADC is administeredat a dosage of between 8 mg/kg and 10 mg/kg.
 15. The method of claim 1,wherein the ADC is administered at a dosage of 10 mg/kg
 16. The methodof claim 1, wherein the treatment results in a reduction in tumor sizeof at least 15%, at least 20%, at least 30%, or at least 40%.
 17. Themethod of claim 1, wherein the ADC comprises 4 or more molecules ofSN-38 conjugated to the antibody or antigen-binding fragment thereof.18. The method of claim 1, wherein the ADC comprises 6 to 8 molecules ofSN-38 conjugated to the antibody or antigen-binding fragment thereof.19. The method of claim 1, wherein the cancer is metastatic.
 20. Themethod of claim 17, further comprising reducing in size or eliminatingthe metastases.
 21. The method of claim 6, wherein the 10-hydroxyposition of SN-38 in MAb-CL2A-SN-38 is a 10-O-ester or 10-O-carbonatederivative using a ‘COR’ moiety, wherein “CO” is carbonyl and the “R”group is selected from (i) an N,N-disubstituted aminoalkyl group“N(CH₃)₂—(CH₂)_(n)—” wherein n is 1-10 and wherein the terminal aminogroup is optionally in the form of a quaternary salt; (ii) an alkylresidue “CH₃—(CH₂)_(n)—” wherein n is 0-10; (iii) an alkoxy moiety“CH₃—(CH₂)n-O—” wherein n is 0-10; (iv) an “N(CH₃)₂—(CH₂)_(n)—O—”wherein n is 2-10; or (v) an “R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” wherein R₁is ethyl or methyl and n is an integer with values of 0-10.
 22. Themethod of claim 1, further comprising administering to the patient atleast one other anti-cancer therapy selected from the group consistingof surgery, external radiation, radioimmunotherapy, immunotherapy,chemotherapy, antisense therapy, interference RNA therapy, treatmentwith a therapeutic agent and gene therapy.
 23. The method of claim 22,wherein the therapeutic agent is a drug, toxin, immunomodulator, secondantibody, antigen-binding fragment of a second antibody, pro-apoptoticagent, toxin, RNase, hormone, radionuclide, anti-angiogenic agent,siRNA, RNAi, chemotherapeutic agent, cytokine, chemokine, prodrug orenzyme.
 24. The method of claim 23, wherein the drug is selected fromthe group consisting of 5-fluorouracil, afatinib, aplidin, azaribine,anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine,bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38,carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide,cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 25. The method ofclaim 23, wherein the wherein the immunomodulator is selected from thegroup consisting of cytokines, lymphokines, monokines, stem cell growthfactors, lymphotoxins, hematopoietic factors, colony stimulating factors(CSF), interferons (IFN), parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, transforming growth factor (TGF), TGF-α,TGF-β, insulin-like growth factor (IGF), erythropoietin, thrombopoietin,tumor necrosis factor (TNF), TNF-α, TNF-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-1 cc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21, IL-23, IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin and endostatin.
 26. The method of claim 23,wherein the radionuclide is selected from the group consisting of ¹¹C,¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru,⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd,¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te, ^(122m)Te, ¹²⁵I,^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho,¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁹⁹Au,²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po, ²¹⁷At,²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁵Ac, ²²⁷Th and ²⁵⁵Fm.
 27. The method of claim 23,wherein the toxin is selected from the group consisting of ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.